Human Eag2

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of Eag2, antibodies to Eag2, methods of detecting Eag2, and methods of screening for modulators of Eag2 potassium channels using biologically active Eag2. The invention further provides, in a computer system, a method of screening for mutations of human Eag2 genes as well as a method for identifying a three-dimensional structure of Eag2 polypeptide monomers.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. S No. 60/143,467, filedJul. 13, 1999, herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The invention provides isolated nucleic acid and amino acidsequences of Eag2, antibodies to Eag2, methods of detecting Eag2, andmethods of screening for modulators of Eag2 potassium channels usingbiologically active Eag2. The invention further provides, in a computersystem, a method of screening for mutations of human Eag2 genes as wellas a method for identifying a three-dimensional structure of Eag2polypeptide monomers.

BACKGROUND OF THE INVENTION

[0004] Potassium channels are involved in a number of physiologicalprocesses, including regulation of heartbeat, dilation of arteries,release of insulin, excitability of nerve cells, and regulation of renalelectrolyte transport. Potassium channels are thus found in a widevariety of animal cells such as nervous, muscular, glandular, immune,reproductive, and epithelial tissue. These channels allow the flow ofpotassium in and/or out of the cell under certain conditions. Forexample, the outward flow of potassium ions upon opening of thesechannels makes the interior of the cell more negative, counteractingdepolarizing voltages applied to the cell. These channels are regulated,e.g., by calcium sensitivity, voltage-gating, second messengers,extracellular ligands, and ATP-sensitivity.

[0005] Potassium channels are made by alpha subunits that fall into 8families, based on predicted structural and functional similarities (Weiet al., Neuropharmacology 35(7):805-829 (1997)). Three of these families(Kv, Eag-related, and KQT) share a common motif of six transmembranedomains and are primarily gated by voltage. Two other families, CNG andSK/IK, also contain this motif but are gated by cyclic nucleotides andcalcium, respectively. The three other families of potassium channelalpha subunits have distinct patterns of transmembrane domains. Slofamily potassium channels, or BK channels have seven transmembranedomains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25):14066-71(1997)) and are gated by both voltage and calcium or pH (Schreiber etal., J. Biol. Chem. 273:3509-16 (1998)). Another family, the inwardrectifier potassium channels (Kir), belong to a structural familycontaining 2 transmembrane domains (see, e.g., Lagrutta et al., Jpn.Heart. J 37:651-660 1996)), and an eighth functionally diverse family(TP, or “two-pore”) contains 2 tandem repeats of this inward rectifiermotif.

[0006] Potassium channels are typically formed by four alpha subunits,and can be homomeric (made of identical alpha subunits) or heteromeric(made of two or more distinct types of alpha subunits). In addition,potassium channels such as those composed of Kv, KQT and Slo or BK alphasubunits have often been found to contain additional, structurallydistinct auxiliary, or beta, subunits. These beta subunits do not formpotassium channels themselves, but instead they act as auxiliarysubunits to modify the functional properties of channels formed by alphasubunits. For example, the Kv beta subunits are cytoplasmic and areknown to increase the surface expression of Kv channels and/or modifyinactivation kinetics of the channel (Heinemann et al., J. Physiol.493:625-633 (1996); Shi et al., Neuron 16(4):843-852 (1996)). In anotherexample, the KQT family beta subunit, minK, primarily changes-activationkinetics (Sanguinetti et al., Nature 384:80-83 (1996)).

[0007] The Kv superfamily of voltage-gated potassium channels includesboth heteromeric and homomeric channels that are typically composed offour subunits. Voltage-gated potassium channels have been found in awide variety of tissues and cell types and are involved in processessuch as neuronal integration, cardiac pacemaking, muscle contraction,hormone section, cell volume regulation, lymphocyte differentiation, andcell proliferation (see, e.g., Salinas et al., J. Biol. Chem.39:24371-24379 (1997)).

[0008] A family of voltage-gated potassium genes, known as the “Eag” orether a go-go family, was identified on the basis of a Drosophilabehavioral mutation with a leg-shaking phenotype (see, e.g., Warmke &Ganetzky, Proc. Nat'l Acad. Sci. USA 91:3438-3442 (1994)). Familymembers from Drosophila and vertebrates have been cloned and fall intothree subfamilies. One such subfamily is called the Eag subfamily and isrepresented, e.g., by Drosophila Eag (Warmke et al., Science252:1560-1562 (1991); Bruggemann et al., Nature 365:445-447 (1993)), andrat, mouse, human, and bovine Eags (Ludwig et al., EMBO J. 13:4451-4458(1994); Robertson et al. Neuropharmacology 35:841-850 (1996); Occhiodoroet al., FEBS Letters 434:177-182 (1998); Shi et al., J. Physiol.115.3:675-682 (1998); Frings et al., J. Gen Physiol. 111:583-599(1998)). A second subfamily, the Erg or “Eag-related gene” family isrepresented, e.g., by human erg (Shi et al., J. Neurosci. 17:9423-9432(1997)). Finally, a third subfamily, the Elk or “Eag-like K⁺ gene” isrepresented, e.g., by Drosophila Elk (Warmke et al., Proc. Natl. Acad.Sci. 91:3438-3442 (1994)).

SUMMARY OF THE INVENTION

[0009] The present invention thus provides for the first time Eag2, apolypeptide monomer that is an alpha subunit of an voltage-gatedpotassium channel. Eag2 has not been previously cloned or identified,and the present invention provides the nucleotide and amino acidsequence of human Eag2.

[0010] In one aspect, the present invention provides an isolated nucleicacid encoding an alpha subunit of a potassium channel, wherein thesubunit: (i) forms, with at least one additional Eag family alphasubunit, a potassium channel having the characteristic of voltagesensitivity; and (ii) comprises an amino acid sequence that has greaterthan about 70% identity to amino acids 720-988 of a human Eag2 aminoacid sequence or comprises an amino acid sequence that has greater thanabout 85% identity to the amino acid sequence of SEQ ID NO:2.

[0011] In another aspect, the present invention provides an isolatednucleic acid that selectively hybridizes under moderately stringenthybridization conditions to a nucleotide sequence of SEQ ID NO: 1.

[0012] In another aspect, the present invention provides an isolatednucleic acid that selectively hybridizes under stringent conditions to anucleotide sequence of SEQ ID NO:1 or to a nucleotide sequence encodingan amino acid sequence of SEQ ID NO:2.

[0013] In another aspect, the present invention provides a method ofdetecting a nucleic acid, by contacting the nucleic acid with a nucleicacid of the invention.

[0014] In another aspect, the present invention provides an isolatedalpha subunit of a potassium channel, wherein the subunit: (i) forms,with at least one additional Eag family alpha subunit, a potassiumchannel having the characteristic of voltage sensitivity; (ii) comprisesan amino acid sequence that has greater than about 70% identity to aminoacids 720-988 of a human Eag2 amino acid sequence or comprises an aminoacid sequence that has greater than about 85% identity to the amino acidsequence of SEQ ID NO:2.

[0015] In one embodiment, the polypeptide specifically binds topolyclonal antibodies generated against SEQ ID NO:2.

[0016] In one embodiment, the nucleic acid encodes human Eag2. Inanother embodiment, the nucleic acid encodes SEQ ID NO:2. In anotherembodiment, the nucleic acid has the nucleotide sequence of SEQ IDNO: 1. In another embodiment, the nucleic acid is amplified by primersthat selectively hybridize under stringent hybridization conditions tothe same sequence as primers selected from the group consisting of:

[0017] ATGCCGGGGGGCAAGAGAGGGCTG (SEQ ID NO:3);

[0018] CTGACCCTAAGCTCATAAGGATGAAC (SEQ ID NO:4);

[0019] CCACCTCATCATCCTGGATGACTTCC (SEQ ID NO:5),

[0020] TTAAAAGTGGATTTCATCTTTGTCAGATTCAGG (SEQ ID NO:6);

[0021] GGGGACCTCATTTACCATGCTGGAG (SEQ ID NO:7); and

[0022] GATTCCCTCATCCACATTTTCAAAGGC (SEQ ID NO:8).

[0023] In another embodiment, the polypeptide monomer has a molecularweight of between about 109 kD and about 119 kD. In another embodiment,the polypeptide monomer has the sequenee of SEQ ID NO:2.

[0024] In another embodiment, the polypeptide monomer comprises an alphasubunit of a heteromeric or homomeric potassium channel.

[0025] In another aspect, the present invention provides an expressionvector comprising the isolated nucleic acid and a host cell transfectedwith such an expression vector.

[0026] In another aspect, the present invention provides an antibodythat selectively binds to the isolated polypeptide monomer.

[0027] In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through apotassium channel, the method comprising the steps of: (i) contactingthe compound with an alpha subunit of a potassium channel, wherein thesubunit: (a) forms, with at least one additional Eag family alphasubunit, a potassium channel having a characteristic of voltagesensitivity; and (b) comprises an amino acid sequence that has greaterthan about 70% to amino acids 720-988 of a human Eag2 amino acidsequence or comprises an amino acid sequence that has greater than about85% identity to the amino acid sequence of SEQ ID NO:2; and (ii)determining the functional effect of the compound upon the potassiumchannel.

[0028] In one embodiment, the functional effect is determined bymeasuring changes in ion flux, current, voltage, or ion concentration.In another embodiment, the polypeptide monomer is recombinant.

[0029] In one embodiment, the functional effect is a physical effect. Inanother embodiment, the functional effect is a chemical effect. Inanother embodiment, the functional effect is determined by measuringligand binding to the channel.

[0030] In one embodiment, the polypeptide is expressed in a cell or cellmembrane, e.g., a eukaryotic cell or a human cell. In anotherembodiment, the polypeptide is attached to a solid support.

[0031] In another aspect, the present invention provides a method ofdetecting the presence of human Eag2 in a biological sample, the methodcomprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a human Eag2-specific reagent thatselectively associates with human Eag2; and, (iii) detecting the levelof human Eag2-specific reagent that selectively associates with thesample.

[0032] In one embodiment, the human Eag 2-specific reagent is selectedfrom the group consisting of: Eag 2-specific antibodies, Eag 2-specificoligonucleotide primers, and Eag 2-specific nucleic acid probes.

[0033] In another aspect, the present invention provides, in a computersystem, a method of screening for mutations of a human Eag2 gene, themethod comprising the steps of: (i) entering into the system at leastabout 50 nucleotides of a first nucleic acid sequence encoding a humanEag2 gene having a nucleotide sequence of SEQ ID NO: 1, andconservatively modified variants thereof; (ii) comparing the firstnucleic acid sequence with a second nucleic acid sequence havingsubstantial identity to the first nucleic acid sequence; and (iii)identifying nucleotide differences between the first and second nucleicacid sequences.

[0034] In one embodiment, the second nucleic acid sequence is associatedwith a disease state. In another embodiment, the step of enteringcomprises entering into the system a nucleotide sequence correspondingto amino acids 720-988 of a human Eag2 gene encoding polypeptide havingan amino acid sequence of SEQ ID NO:2.

[0035] In another aspect, the present invention provides a method foridentifying a compound that increases or decreases ion flux through apotassium channel comprising an Eag2 polypeptide, the method comprisingthe steps of: (i) entering into a computer system an amino acid sequenceof at least 50 amino acids of an Eag2 polypeptide or at least 150nucleotides of a nucleic acid encoding the Eag2 polypeptide, the Eag2polypeptide comprising a subsequence having at least 70% amino acidsequence to amino acids 720 to 988 of SEQ ID NO:2 or comprises an aminoacid sequence that has greater than about 85% identity to the amino acidsequence of SEQ ID NO:2; (ii) generating a three-dimensional structureof the polypeptide encoded by the amino acid sequence; (iii) generatinga three-dimensional structure of the potassium channel comprising theEag2 polypeptide; (iv) generating a three-dimensional structure of thecompound; and (v) comparing the three-dimensional structures of thepolypeptide and the compound to determine whether or not the compoundbinds to the polypeptide.

[0036] In another aspect, the present invention provides a method ofmodulating ion flux through an Eag potassium channel to treat disease ina subject, the method comprising the step of administering to thesubject a therapeutically effective amount of a compound identifiedusing the methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1: Amino acid alignment of human Eag2 and human Eag1.Identical amino acids are shaded. Amino acid positions are given at theleft margin. The region of highest homology extends from the aminoterminus through amino acid 720 of the Eag2 sequence. This regionincludes the “core” of the channel, six transmembrane domains and aP-region that contributes to the channel pore, and a putativecyclic-nucleotide binding domain in the C-terminal cytoplasmic region.Within this region, Eag2 and Eag1 share 85% amino acid identity. Thehomology drops off significantly from this region to the C-terminus(amino acids 720-998 of Eag2).

[0038]FIG. 2: Functional expression of Eag2 in Xenopus oocytes. A.)Families of outward currents recorded from an oocyte injected with Eag2cRNA. Voltage steps ranged from −80 mV to +40 mV in 10 mV increments;the holding potential was −90 mV and tail currents were elicited at −60mV. B.) A normalized conductance vs. voltage curve for the Eag2 currentshown in A. The line shows a bolztmann fit to the data, and gives ahalf-activation voltage (V₅₀) of −28.5 mV. Note the low slope of thecurve and very hyperpolarized activation of the current. C.) Sensitivityof the activation rate of Eag2 to holding potential. Eag2 currents wereactivated by a step to 0 mV from holding potentials that varied from−120 mV to −40 mV in 20 mV increments. Activation had both fast and slowcomponents, with the slow component dominating at hyperpolarized holdingpotentials and the fast component dominating at holding potentials neartypical cellular resting potentials.

[0039]FIG. 3: Transient expression of Eag2 in Chinese hamster ovarycells. A.) Families of outward currents recorded by whole patch clampfrom a CHO cell transfected with Eag2. The holding potential was −90 mVand voltage steps ranged from −100 mV to +40 mV in 10 mV increments.Tail currents were measured at −120 mV. B.) A normalized conductance vs.voltage curve for the currents shown in A. The line shows a boltzmannfit of the data, and gives a V₅₀ of-21 mV. The V₅₀ and slope are similarto that found for Eag2 expression in Xenopus oocytes.

DETAILED DESCRIPTION OF THE INVENTION

[0040] I. Introduction

[0041] The present invention provides for the first time a nucleic acidencoding Eag2, identified and cloned from human tissue. This polypeptidemonomer is a member of the “Kv” superfamily of potassium channelmonomers and the “Eag” (ether a go-go) family and subfamily of potassiumchannel monomers. Members of this family are polypeptide monomers thatare subunits of voltage-gated potassium channels having sixtransmembrane regions (K=potassium, v=voltage-gated). Voltage-gatedpotassium channels have significant roles in maintaining the restingpotential and in controlling excitability of a cell.

[0042] The invention also provides methods of screening for activatorsand inhibitors of voltage-gated potassium channels that contain an Eag2subunit. Such modulators of voltage-gated channel activity are usefulfor treating disorders involving abnormal ion flux, e.g., CNS disorderssuch as migraines, hearing and vision problems, Alzheimer's disease,learning and memory disorders, seizures, psychotic disorders, and asneuroprotective agents (e.g., to prevent stroke).

[0043] Furthermore, the invention provides assays for Eag2 activitywhere Eag2 acts as a direct or indirect reporter molecule. Such uses ofEag2 as a reporter molecule in assay and detection systems have broadapplications, e.g., Eag2 can be used as a reporter molecule to measurechanges in potassium concentration, membrane potential, current flow,ion flux, transcription, signal transduction, receptor-ligandinteractions, second messenger concentrations, in vitro, in vivo, and exvivo. In one embodiment, Eag2 can be used as an indicator of currentflow in a particular direction (e.g., outward or inward potassium flow),and in another embodiment, Eag2 can be used as an indirect reporter viaattachment to a second reporter molecule such as green fluorescentprotein.

[0044] The invention also provides for methods of detecting Eag2 nucleicacid and protein expression, allowing investigation of the channeldiversity provided by hEag2, as well as diagnosis of disease caused byabnormal ion flux, e.g., CNS disorders such as migraines, hearing andvision problems, seizures, and psychotic disorders.

[0045] Finally, the invention provides for a method of screening formutations of Eag2 genes or proteins. The invention includes, but is notlimited to, methods of screening for mutations in Eag2 with the use of acomputer. Similarly, the invention provides for methods of identifyingthe three-dimensional structure of Eag2, as well as the resultingcomputer readable images or data that comprise the three dimensionalstructure of Eag2. Other methods for screening for mutations of Eag2genes or proteins include high density oligonucleotide arrays, PCR,immunoassays and the like.

[0046] Functionally, Eag2 is an alpha subunit of an voltage-gatedpotassium channel. Typically, such voltage-gated channels areheteromeric or homomeric and contain four alpha subunits or monomerseach with six transmembrane domains. Heteromeric Eag channels cancomprise one or more Eag2 alpha subunits along with one or moreadditional alpha subunits from the Eag family, preferably from the Eagsubfamily, such as Eag1. Eag2 channels may also be homomeric. Inaddition, such channels may comprise one or more auxiliary betasubunits. The presence of Eag2 in an voltage-gated potassium channel mayalso modulate the activity of the heteromeric channel and thus enhancechannel diversity. Channel diversity is also enhanced with alternativelyspliced forms of Eag2.

[0047] Structurally, the nucleotide sequence of human Eag2 (SEQ IDNO: 1) encodes a polypeptide monomer of approximately 988 amino acidswith a predicted molecular weight of approximately 114 kDa (SEQ ID NO:2)and a predicted range of 109-119 kDa. In particular, the amino acidsequence of hEag2 has an “C-terminal” region (approximately amino acids720 to the end of the amino acid sequence, see, e.g., amino acids720-988 of SEQ ID NO:2, hEag2) that distinguishes Eag2 from other Eagfamily members. Related Eag2 genes from other species share at leastabout 70%, preferably 75, 80, 85, 90, or 95% amino acid identity in thisregion. Furthermore, related Eag2 genes from other species share about85% identity to the amino acid sequence of SED ID NO:2.

[0048] The present invention also provide polymorphic variants of thehEag2 depicted in SEQ ID NO:2: variant #1, in which an isoleucineresidue is substituted for the valine residue at amino acid position973; variant #2, in which a lysine residue is substituted for thearginine residue at amino acid position 927; variant #3, in which analanine residue is substituted for the threonine residue at amino acidposition 905, and variant #4, in which an isoleucine residue issubstituted for the valine residue at amino acid position 40.

[0049] Specific regions of the hEag2 nucleotide and amino acid sequencemay be used to identify polymorphic variants, interspecies homologs, andalleles of hEag2. This identification can be made in vitro, e.g., understringent hybridization conditions and sequencing, or by using thesequence information in a computer system for comparison with othernucleotide sequences, or by using antibodies raised against hEag2.Typically, identification of polymorphic variants and alleles of hEag2is made by comparing the amino acid sequence (or the nucleic acidsequence encoding the nucleic acid sequence) of the “C-terminal region”(approximately amino acids 720-988 of hEag2, see SEQ ID NO:2 forexample). Amino acid identity of approximately at least 70% or above,preferably 75%, 80%, or 85%, most preferably 90-95% or above in theC-terminal region typically demonstrates that a protein is a polymorphicvariant, interspecies homolog, or allele of hEag2. Alternatively, aminoacid sequence identity of at least 85% or above to the amino acidsequence of SEQ ID NO:2 typically demonstrates that a protein is apolymorphic variant, interspecies homolog, or allele of hEag2. Sequencecomparison can be performed using any of the sequence comparisonalgorithms discussed below. Antibodies that bind specifically to thesubunit association region of hEag2 can also be used to identifyalleles, interspecies homologs, and polymorphic variants.

[0050] Polymorphic variants, interspecies homologs, and alleles of hEag2can be confirmed by expressing or co-expressing the putative Eag2polypeptide monomer and examining whether it forms a potassium channelwith Eag2/Eag functional characteristics, such as voltage-gating. Thisassay is used to demonstrate that a protein having about 70% or greater,preferably 75%, 80%, 85%, 90%, or 95% or greater amino acid identity tothe “C-terminal” region of hEag2 shares the same functionalcharacteristics as hEag2 and is therefore a species of hEag2. This assayis also used to demonstrate that a protein having about 85% or greater,preferably 90%, or 95% or greater amino acid identity to the amino acidsequence of SEQ ID NO:2 shares the same functional characteristics ashEag2 and is therefore a species of hEag2. Typically, hEag2 having theamino acid sequence of SEQ ID NO:2 is used as a positive control incomparison to the putative Eag2 protein to demonstrate theidentification of a polymorphic variant, interspecies homolog, or alleleof hEag2.

[0051] Eag2 nucleotide and amino acid sequence information may also beused to construct models of voltage-gated potassium channels in acomputer system. These models are subsequently used to identifycompounds that can activate or inhibit voltage-gated potassium channelscomprising Eag2. Such compounds that modulate the activity of channelscomprising Eag2 can be used to investigate the role of Eag2 inmodulation of channel activity and in channel diversity.

[0052] The isolation of biologically active Eag2 for the first timeprovides a means for assaying for inhibitors and activators ofvoltage-gated potassium channels that comprise Eag2 subunits.Biologically active Eag2 is useful for testing inhibitors and activatorsof voltage-gated potassium channels comprising subunits of Eag2 andother Eag family members using in vivo and in vitro expression thatmeasure, e.g., changes in ion flux, ion concentration, voltage, current,or ligand binding. Such activators and inhibitors identified using anvoltage-gated potassium channel comprising at least one Eag2 subunit,preferably four Eag2 subunits, can be used to further study voltagegating, channel kinetics and conductance properties of potassiumchannels. Such activators and inhibitors are useful as pharmaceuticalagents for treating diseases involving abnormal ion flux, e.g., CNSdisorders, as described above. Methods of detecting Eag2 and expressionof channels comprising Eag2 are also useful for diagnostic applicationsfor diseases involving abnormal ion flux, e.g., CNS disorders and otherdisorders. For example, chromosome localization of the gene encodinghuman Eag2 can be used to identify diseases caused by and associatedwith hEag2. Methods of detecting Eag2 are also useful for examining therole of Eag2 in channel diversity and modulation of channel activity.

[0053] II. Definitions

[0054] As used herein, the following terms have the meanings ascribed tothem unless specified otherwise.

[0055] The phrase “voltage-gated” activity or “voltage-gating” refers toa characteristic of a potassium channel composed of individualpolypeptide monomers or subunits. Generally, the probability of avoltage-gated potassium channel opening increases as a cell isdepolarized. Voltage-gated potassium channels primarily allow efflux ofpotassium because they have greater probabilities of being open atmembrane potentials more positive than the membrane potential forpotassium (EK) in typical cells. EK, or the membrane potential forpotassium, depends on the relative concentrations of potassium foundinside and outside the cell membrane, and is typically between −60 and−100 mV for mammalian cells. EK is the membrane potential at which thereis no net flow of potassium ion because the electrical potential (i.e.,voltage potential) driving potassium influx is balanced by theconcentration gradient (the [K⁺] potential) directing potassium efflux.This value is also known as the “reversal potential” or the “Nernst”potential for potassium. Some voltage-gated potassium channels undergoinactivation, which can reduce potassium efflux at higher membranepotentials. Potassium channels can also allow potassium influx incertain instances when they remain open at membrane potentials negativeto E_(K) (see, e.g., Adams & Normer, in Potassium Channels, pp. 40-60(Cook, ed., 1990)). The characteristic of voltage gating can be measuredby a variety of techniques for measuring changes in current flow and ionflux through a channel, e.g., by changing the [K⁺] of the externalsolution and measuring the activation potential of the channel current(see, e.g., U.S. Pat. No. 5,670,335), by measuring current with patchclamp techniques or voltage clamp under different conditions, and bymeasuring ion flux with radiolabeled tracers or voltage-sensitive dyesunder different conditions.

[0056] “Homomeric channel” refers to an Eag2 channel composed ofidentical alpha subunits, whereas “heteromeric channel” refers to an Eagchannel composed of at least one Eag2 alpha subunit plus at least oneother different type of alpha subunit from a related gene family such asthe Eag family, preferably from the Eag subfamily, e.g., Eag1. Bothhomomeric and heteromeric channels can include auxiliary beta subunits.Typically, the channel is composed of four alpha subunits and thechannel can be heteromeric or homomeric.

[0057] A “beta subunit” is a polypeptide monomer that is an auxiliarysubunit of a potassium channel composed of alpha subunits; however, betasubunits alone cannot form a channel (see, e.g., U.S. Pat. No.5,776,734). Beta subunits are known, for example, to increase the numberof channels by helping the alpha subunits reach the cell surface, changeactivation kinetics, and change the sensitivity of natural ligandsbinding to the channels. Beta subunits can be outside of the pore regionand associated with alpha subunits comprising the pore region. They canalso contribute to the external mouth of the pore region.

[0058] The phrase “C-terminal region” refers to the region of Eag2 thatstructurally identifies this particular protein (approximately aminoacids 720-988 of hEag2, see SEQ ID NO:2). This region can be used toidentify Eag2 polymorphic variants and Eag2 alleles of hEag2, throughamino acid sequence identity comparison using a sequence comparisonalgorithm such as BLASTP. “Eag2” refers to a polypeptide that is asubunit or monomer of an voltage-gated potassium channel, a member ofthe Eag family and subfamily, and a member of the Kv superfamily ofpotassium channel monomers. When Eag2 is part of a potassium channel,either a homomeric or heteromeric potassium channel, the channel hasvoltage-gated activity. The term Eag2 therefore refers to polymorphicvariants, alleles, mutants, and interspecies homologs that: (1) have aC-terminal region that has greater than about 70% amino acid sequenceidentity, preferably about 75, 80, 85, 90, or 95% amino acid sequenceidentity, to a hEag2 C-terminal region or comprises an amino acidsequence that has greater than about 85% identity to the amino acidsequence of SEQ ID NO:2; (2) bind to antibodies raised against animmunogen comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, amino acids 720-988 of SEQ ID NO:2, andconservatively modified variants thereof; (3) specifically hybridizeunder stringent hybridization conditions to a sequence selected from thegroup consisting of SEQ ID NO: 1, the nucleotide sequence encoding aminoacids 720-988 of SEQ ID NO:2, and conservatively modified variantsthereof; or (4) are amplified by primers that specifically hybridizeunder stringent hybridization conditions to the same sequence as aprimer set consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0059] The phrase “functional effects” in the context of assays fortesting compounds affecting a channel comprising Eag2 includes thedetermination of any parameter that is indirectly or directly under theinfluence of the channel. It includes changes in ion flux and membranepotential, changes in ligand binding, and also includes otherphysiologic effects such as increases or decreases of transcription orhormone release.

[0060] “Determining the functional effect” refers to examining theeffect of a compound that increases or decreases ion flux on a cell orcell membrane in terms of cell and cell membrane function. The ion fluxcan be any ion that passes through a channel and analogues thereof,e.g., potassium, rubidium, sodium. Preferably, the term refers to thefunctional effect of the compound on the channels comprising hEag2,e.g., changes in ion flux including radioisotopes, current amplitude,ligand binding, membrane potential, current flow, transcription, proteinbinding, phosphorylation, dephosphorylation, second messengerconcentrations (cAMP, cGMP, Ca²⁺, IP₃) and other physiological effectssuch as hormone and neurotransmitter release, as well as changes involtage and current. Such functional effects can be measured by anymeans known to those skilled in the art, e.g., patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, and the like.

[0061] “Inhibitors,” “activators” or “modulators” of voltage-gatedpotassium channels comprising hEag2 refer to inhibitory or activatingmolecules identified using in vitro and in vivo assays for hEag2 channelfunction. Inhibitors are compounds that decrease, block, prevent, delayactivation, inactivate, desensitize, or down regulate the channel.Activators are compounds that increase, open, activate, facilitate,enhance activation, sensitize or up regulate channel activity. Suchassays for inhibitors and activators include e.g., expressing hEag2 incells or cell membranes and then measuring flux of ions through thechannel and determining changes in polarization (i.e., electricalpotential). Alternatively, cells expressing endogenous hEag2 channelscan be used in such assays. To examine the extent of inhibition, samplesor assays comprising an hEag2 channel are treated with a potentialactivator or inhibitor and are compared to control samples without theinhibitor. Control samples (untreated with inhibitors) are assigned arelative hEag2 activity value of 100%. Inhibition of channels comprisinghEag2 is achieved when the hEag2 activity value relative to the controlis about 90%, preferably 50%, more preferably 25-0%. Activation ofchannels comprising hEag2 is achieved when the hEag2 activity valuerelative to the control is 110%, more preferably 150%, most preferablyat least 200-500% higher or 1000% or higher.

[0062] “Biologically active” hEag2 refers to hEag2 that has the abilityto form a potassium channel having the characteristic of voltage-gatingtested as described above.

[0063] The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated hEag2 nucleic acid is separated from openreading frames that flank the hEag2 gene and encode proteins other thanhEag2. The term “purified” denotes that a nucleic acid or protein givesrise to essentially one band in an electrophoretic gel. Particularly, itmeans that the nucleic acid or protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

[0064] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0065] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0066] A particular nucleic acid sequence also implicitly encompasses“splice variants.” Similarly, a particular protein encoded by a nucleicacid implicitly encompasses any protein encoded by a splice variant ofthat nucleic acid. “Splice variants,” as the name suggests, are productsof alternative splicing of a gene. After transcription, an initialnucleic acid transcript may be spliced such that different (alternate)nucleic acid splice products encode different polypeptides. Mechanismsfor the production of splice variants vary, but include alternatesplicing of exons. Alternate polypeptides derived from the same nucleicacid by read-through transcription are also encompassed by thisdefinition. Any products of a splicing reaction, including recombinantforms of the splice products, are included in this definition. Anexample of potassium channel splice variants is discussed in Leicher, etal., J. Biol. Chem. 273(52):35095-35101 (1998).

[0067] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

[0068] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid; but that functions in amanner similar to a naturally occurring amino acid.

[0069] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0070] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

[0071] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0072] The following eight groups each contain amino acids that areconservative substitutions for one another:

[0073] 1) Alanine (A), Glycine (G);

[0074] 2) Aspartic acid (D), Glutamic acid (E);

[0075] 3) Asparagine (N), Glutamine (Q);

[0076] 4) Arginine (R), Lysine (K);

[0077] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0078] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0079] 7) Serine (S), Threonine (T); and

[0080] 8) Cysteine (C), Methionine (M)

[0081] (see, e.g., Creighton, Proteins (1984)).

[0082] Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I: The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofpolypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

[0083] The subunits and potassium channels of the invention comprisedomain such as a “pore” domain. These domains can be structurallyidentified using methods known to those of skill in the art, such assequence analysis programs that identify hydrophobic and hydrophilicdomains (see, e.g., Kyte & Doolittle, J. Mol. Biol. 157:105-132 (1982)).Such domains are useful for making chimeric proteins and for in vitroassays of the invention.

[0084] A “label” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include ³²P, fluorescent dyes, electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, or haptens and proteins for which antisera or monoclonalantibodies are available (e.g., the polypeptide of SEQ ID NO:2 can bemade detectable, e.g., by incorporating a radiolabel into the peptide,and used to detect antibodies specifically reactive with the peptide).

[0085] As used herein a “nucleic acid probe or oligonucleotide” isdefined as a nucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

[0086] A “labeled nucleic acid probe or oligonucleotide” is one that isbound, either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

[0087] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0088] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

[0089] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0090] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0091] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity overa specified region such as the hEag2 C-terminal region or 85% identityor more to the amino acid sequence of SEQ ID NO:2), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Suchsequences are then said to be “substantially identical.” This definitionalso refers to the compliment of a test sequence. Preferably, theidentity exists over a region that is at least about 50 amino acids ornucleotides in length, or more preferably over a region that is 75-100amino acids or nucleotides in length.

[0092] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters. For sequencecomparison of nucleic acids and proteins to Eag2 nucleic acids andproteins, e.g., hEag2, the BLAST and BLAST 2.0 algorithms and thedefault parameters discussed below are used.

[0093] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

[0094] A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

[0095] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0096] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

[0097] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0098] The phrase “stringent hybridization conditions” refers toconditions under which a probe will hybridize to its target subsequence,typically in a complex mixture of nucleic acid, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For high stringencyhybridization, a positive signal is at least two times background,preferably 10 times background hybridization. Exemplary high stringencyor stringent hybridization conditions include: 50% formamide, 5×SSC and1% SDS incubated at 42° C. or 5×SSC and 1% SDS incubated at 65° C., witha wash in 0.2×SSC and 0.1% SDS at 65° C.

[0099] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cased, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

[0100] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0101] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0102] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

[0103] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0104] An “anti-hEag2” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the hEag2 gene, cDNA, or asubsequence thereof.

[0105] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0106] The term “immunoassay” is an assay that uses an antibody tospecifically bind an antigen. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen.

[0107] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to hEag2, encoded in SEQ IDNO:2, splice variants, or portions thereof, can be selected to obtainonly those polyclonal antibodies that are specifically immunoreactivewith hEag2 and not with other proteins, except for polymorphic variantsand alleles of hEag2. This selection may be achieved by subtracting outantibodies that cross-react with molecules such as rat Eag1, DrosophilaEag, mouse Eag1, and human Eag1. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

[0108] The phrase “selectively associates with” refers to the ability ofa nucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

[0109] By “host cell” is meant a cell that contains an expression vectorand supports the replication or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells such as CHO, HeLaand the like, e.g., cultured cells, explants, and cells in vivo.

[0110] “Biological sample” as used herein is a sample of biologicaltissue or fluid that contains hEag2 or nucleic acid encoding hEag2protein. Such samples include, but are not limited to, tissue isolatedfrom humans. Biological samples may also include sections of tissuessuch as frozen sections taken for histologic purposes. A biologicalsample is typically obtained from a eukaryotic organism, preferablyeukaryotes such as fungi, plants, insects, protozoa, birds, fish,reptiles, and preferably a mammal such as rat, mice, cow, dog, guineapig, or rabbit, and most preferably a primate such as chimpanzees orhumans.

[0111] III. Isolating the Gene Encoding Eag2

[0112] A. General Recombinant DNA Methods

[0113] This invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

[0114] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

[0115] Oligonucleotides that are not commercially available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22:1859-1862 (1981), using an automated synthesizer, as describedin Van Devanter et. al., Nucleic Acids Res. 12:6159-6168 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255:137-149 (1983).

[0116] The sequence of the cloned genes and synthetic oligonucleotidescan be verified after cloning using, e.g., the chain termination methodfor sequencing double-stranded templates of Wallace et al., Gene16:21-26 (1981).

[0117] B. Cloning Methods for the Isolation of Nucleotide SequencesEncoding hEag2

[0118] In general, the nucleic acid sequences encoding Eag2 and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. For example, hEag2 sequences are typicallyisolated from human nucleic acid (genomic or cDNA) libraries byhybridizing with a nucleic acid probe or polynucleotide, the sequence ofwhich can be derived from SEQ ID NO:1, optionally from the regionencoding the C-terminal region. A suitable tissue from which Eag2 RNAand cDNA can be isolated is brain tissue, e.g., whole brain orhippocampus.

[0119] Amplification techniques using primers can also be used toamplify and isolate Eag2 from DNA or RNA. The following primers can alsobe used to amplify a sequence of hEag2: ATGCCGGGGGGCAAGAGAGGGCTG (SEQ IDNO:3), CTGACCCTAAGCTCATAAGGATGAAC (SEQ ID NO:4)CCACCTCATCATCCTGGATGACTTCC (SEQ ID NO:5),TTAAAAGTGGATTTCATCTTTGTCAGATTCAGG (SEQ ID NO:6)GGGGACCTCATTTACCATGCTGGAG (SEQ ID NO:7) and GATTCCCTCATCCACATTTTCAAAGGC(SEQ ID NO:8). These primers can be used, e.g., to amplify either thefull length sequence or a probe of one to several hundred nucleotides,which is then used to screen a human library for full-length hEag2.

[0120] Nucleic acids encoding Eag2 can also be isolated from expressionlibraries using antibodies as probes. Such polyclonal or monoclonalantibodies can be raised using the sequence of SEQ ID NO:2.

[0121] Human Eag2 polymorphic variants and alleles that aresubstantially identical to the C-terminal region of hEag2 can beisolated using hEag2 nucleic acid probes and oligonucleotides understringent hybridization conditions, by screening libraries.Alternatively, expression libraries can be used to clone Eag2 and Eag2polymorphic variants and alleles by detecting expressed homologsimmunologically with antisera or purified antibodies made against hEag2or portions thereof (e.g., the C-terminal region of hEag2), which alsorecognize and selectively bind to the Eag2 homolog.

[0122] To make a cDNA library, one should choose a source that is richin Eag2 mRNA, e.g., tissue such as brain, e.g., whole brain orhippocampus. The mRNA is then made into cDNA using reversetranscriptase, ligated into a recombinant vector, and transfected into arecombinant host for propagation, screening and cloning. Methods formaking and screening cDNA libraries are well known (see, e.g., Gubler &Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al.,supra).

[0123] For a genomic library, the DNA is extracted from the tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

[0124] An alternative method of isolating Eag2 nucleic acid and itshomologs combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of Eag2 directly from mRNA, from cDNA, from genomic librariesor cDNA libraries. Degenerate oligonucleotides can be designed toamplify hEag2 homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of Eag2 encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

[0125] Gene expression of Eag2 can also be analyzed by techniques knownin the art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, high density polynucleotidearray technology and the like.

[0126] Synthetic oligonucleotides can be used to construct recombinantEag2 genes for use as probes or for expression of protein. This methodis performed using a series of overlapping oligonucleotides usually40-120 bp in length, representing both the sense and nonsense strands ofthe gene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the Eag2 gene. The specificsubsequence is then ligated into an expression vector.

[0127] The gene for Eag2 is typically cloned into intermediate vectorsbefore transformation into prokaryotic or eukaryotic cells forreplication and/or expression. These intermediate vectors are typicallyprokaryote vectors, e.g., plasmids, or shuttle vectors.

[0128] C. Expression in Prokaryotes and Eukaryotes

[0129] To obtain high level expression of a cloned gene, such as thosecDNAs encoding Eag2, one typically subclones Eag2 into an expressionvector that contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al., supra. Bacterial expressionsystems for expressing the Eag2 protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available.

[0130] Selection of the promoter used to direct expression of aheterologous nucleic acid depends on the particular application. Thepromoter is preferably positioned about the same distance from theheterologous transcription start site as it is from the transcriptionstart site in its natural setting. As is known in the art, however, somevariation in this distance can be accommodated without loss of promoterfunction.

[0131] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the Eag2 encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding Eag2and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

[0132] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0133] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

[0134] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the CMV promoter, SV40early promoter, SV40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

[0135] Expression of proteins from eukaryotic vectors can be also beregulated using inducible promoters. With inducible promoters,expression levels are tied to the concentration of inducing agents, suchas tetracycline or ecdysone, by the incorporation of response elementsfor these agents into the promoter. Generally, high level expression isobtained from inducible promoters only in the presence of the inducingagent; basal expression levels are minimal. Inducible expression vectorsare often chosen if expression of the protein of interest is detrimentalto eukaryotic cells.

[0136] Some expression systems have markers that provide geneamplification such as thymidine kinase and dihydrofolate reductase.Alternatively, high yield expression systems not involving geneamplification are also suitable, such as using a baculovirus vector ininsect cells, with a Eag2 encoding sequence under the direction of thepolyhedrin promoter or other strong baculovirus promoters.

[0137] The elements that are typically included in expression vectorsalso include a replicon that functions in E. coli, a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Theparticular antibiotic resistance gene chosen is not critical, any of themany resistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

[0138] Standard transfection methods are used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofEag2 protein, which are then purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

[0139] Any of the well-known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, liposomes, microinjection, plasma vectors, viralvectors and any of the other well known methods for introducing clonedgenomic DNA, cDNA, synthetic DNA or other foreign genetic material intoa host cell (see, e.g., Sambrook et al., supra). It is only necessarythat the particular genetic engineering procedure used be capable ofsuccessfully introducing at least one gene into the host cell capable ofexpressing Eag2.

[0140] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofEag2, which is recovered from the culture using standard techniquesidentified below.

[0141] IV. Purification of Eag2 Polypeptides

[0142] Either naturally occurring or recombinant Eag2 can be purifiedfor use in functional assays. Naturally occurring Eag2 monomers can bepurified, e.g., from human tissue such as whole brain or hippocampus,and any other source of a Eag2 homolog. Recombinant Eag2 monomers can bepurified from any suitable expression system.

[0143] The Eag2 monomers may be purified to substantial purity bystandard techniques, including selective precipitation with suchsubstances as ammonium sulfate; column chromatography,immunopurification methods, and others (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook et al., supra).

[0144] A number of procedures can be employed when recombinant Eag2monomers are being purified. For example, proteins having establishedmolecular adhesion properties can be reversible fused to the Eag2monomers. With the appropriate ligand, the Eag2 monomers can beselectively adsorbed to a purification column and then freed from thecolumn in a relatively pure form. The fused protein is then removed byenzymatic activity. Finally the Eag2 monomers could be purified usingimmunoaffinity columns.

[0145] A. Purification of Eag2 Monomers from Recombinant Bacteria

[0146] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

[0147] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofthe Eag2 monomers inclusion bodies. For example, purification ofinclusion bodies typically involves the extraction, separation and/orpurification of inclusion bodies by disruption of bacterial cells, e.g.,by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mMMgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can belysed using 2-3 passages through a French Press, homogenized using aPolytron (Brinkman Instruments) or sonicated on ice. Alternate methodsof lysing bacteria are apparent to those of skill in the art (see, e.g.,Sambrook et al., supra; Ausubel et al., supra).

[0148] If necessary, the inclusion bodies are solubilized, and the lysedcell suspension is typically centrifuged to remove unwanted insolublematter. Proteins that formed the inclusion bodies may be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. Other suitable buffers are known to those skilled in the art.Human Eag2 monomers are separated from other bacterial proteins bystandard separation techniques, e.g., with Ni-NTA agarose resin.

[0149] Alternatively, it is possible to purify the Eag2 monomers frombacteria periplasm. After lysis of the bacteria, when the Eag2 monomersare exported into the periplasm of the bacteria, the periplasmicfraction of the bacteria can be isolated by cold osmotic shock inaddition to other methods known to skill in the art. To isolaterecombinant proteins from the periplasm, the bacterial cells arecentrifuged to form a pellet. The pellet is resuspended in a buffercontaining 20% sucrose. To lyse the cells, the bacteria are centrifugedand the pellet is resuspended in ice-cold 5 mM MgSO₄ and kept in an icebath for approximately 10 minutes. The cell suspension is centrifugedand the supernatant decanted and saved. The recombinant proteins presentin the supernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

[0150] B. Standard Protein Separation Techniques for Purifying the Eag2Monomers

[0151] Solubility Fractionation

[0152] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0153] Size Differential Filtration

[0154] The molecular weight of the Eag2 monomers can be used to isolatedit from proteins of greater and lesser size using ultrafiltrationthrough membranes of different pore size (for example, Amicon orMillipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

[0155] Column Chromatography

[0156] The Eag2 monomers can also be separated from other proteins onthe basis of its size, net surface charge, hydrophobicity, and affinityfor ligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

[0157] V. Immunological Detection of Eag2

[0158] In addition to the detection of Eag2 genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect the Eag2 monomers. Immunoassays can be used toqualitatively or quantitatively analyze the Eag2 monomers. A generaloverview of the applicable technology can be found in Harlow & Lane,Antibodies: A Laboratory Manual (1988).

[0159] A. Antibodies to Eag2 Monomers

[0160] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with the Eag2 monomers are known to those of skill inthe art (see, e.g., Coligan, Current Protocols in Immunology (1991);Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles andPractice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497(1975). Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).

[0161] A number of immunogens comprising portions of Eag2 monomers maybe used to produce antibodies specifically reactive with Eag2 monomers.For example, recombinant Eag2 monomers or an antigenic fragment thereof,such as the C-terminal region, can be isolated as described herein.Recombinant protein can be expressed in eukaryotic or prokaryotic cellsas described above, and purified as generally described above.Recombinant protein is the preferred immunogen for the production ofmonoclonal or polyclonal antibodies. Alternatively, a synthetic peptidederived from the sequences disclosed herein and conjugated to a carrierprotein can be used an immunogen. Naturally occurring protein may alsobe used either in pure or impure form. The product is then injected intoan animal capable of producing antibodies. Either monoclonal orpolyclonal antibodies may be generated, for subsequent use inimmunoassays to measure the protein.

[0162] Methods of production of polyclonal antibodies are known to thoseof skill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see Harlow & Lane, supra).

[0163] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989).

[0164] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 10⁴ or greaterare selected and tested for their cross reactivity against non-Eag2proteins, other Eag1 orthologs such as human Eag1 or related subfamilymembers such as rat Eag2), using a competitive binding immunoassay.Specific polyclonal antisera and monoclonal antibodies will usually bindwith a K_(d) of at least about 0.1 mM, more usually at least about 1 μM,preferably at least about 0.1 μM or better, and most preferably, 0.01 μMor better.

[0165] Once the specific antibodies against a Eag2 are available, theEag2 can be detected by a variety of immunoassay methods. For a reviewof immunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

[0166] B. Immunological Binding Assays

[0167] The Eag2 can be detected and/or quantified using any of a numberof well recognized immunological binding assays (see, e.g., U.S. Pat.Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review ofthe general immunoassays, see also Methods in Cell Biology: Antibodiesin Cell Biology, volume 37 (Asai, ed. 1993); Basic and ClinicalImmunology (Stites & Terr, eds., 7^(th) ed. 1991). Immunological bindingassays (or immunoassays) typically use an antibody that specificallybinds to a protein or antigen of choice (in this case the Eag2 or anantigenic subsequence thereof). The antibody (e.g., anti-Eag2) may beproduced by any of a number of means well known to those of skill in theart and as described above.

[0168] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeled Eag2polypeptide or a labeled anti-Eag2 antibody. Alternatively, the labelingagent may be a third moiety, such a secondary antibody, whichspecifically binds to the antibody/Eag2 complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). Thelabeling agent can be modified with a detectable moiety, such as biotin,to which another molecule can specifically bind, such as streptavidin. Avariety of detectable moieties are well known to those skilled in theart.

[0169] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0170] Non-Competitive Assay Formats

[0171] Immunoassays for detecting the Eag2 in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-Eag2 subunit antibodies can bebound directly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture Eag2 present in the test sample. TheEag2 monomers are thus immobilized and then bound by a labeling agent,such as a second Eag2 antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

[0172] Competitive Assay Formats

[0173] In competitive assays, the amount of the Eag2 present in thesample is measured indirectly by measuring the amount of known, added(exogenous) Eag2 displaced (competed away) from an anti-Eag2 antibody bythe unknown Eag2 present in a sample. In one competitive assay, a knownamount of the Eag2 is added to a sample and the sample is then contactedwith an antibody that specifically binds to the Eag2. The amount ofexogenous Eag2 bound to the antibody is inversely proportional to theconcentration of the Eag2 present in the sample. In a particularlypreferred embodiment, the antibody is immobilized on a solid substrate.The amount of Eag2 bound to the antibody may be determined either bymeasuring the amount of Eag2 present in a Eag2/antibody complex, oralternatively by measuring the amount of remaining uncomplexed protein.The amount of Eag2 may be detected by providing a labeled Eag2 molecule.

[0174] A hapten inhibition assay is another preferred competitive assay.In this assay the known Eag2 is immobilized on a solid substrate. Aknown amount of anti-Eag2 antibody is added to the sample, and thesample is then contacted with the immobilized Eag2. The amount ofanti-Eag2 antibody bound to the known immobilized Eag2 is inverselyproportional to the amount of Eag2 present in the sample. Again, theamount of immobilized antibody may be detected by detecting either theimmobilized fraction of antibody or the fraction of the antibody thatremains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

[0175] Cross-Reactivity Determinations

[0176] Immunoassays in the competitive binding format can also be usedfor crossreactivity determinations for Eag2. For example, a protein atleast partially encoded by SEQ ID NO:2 or an immunogenic region thereof,such as the C-terminal region (amino acids 720-988), can be immobilizedto a solid support. Other proteins such as other Eag subfamily memberssuch as any mammalian Eag1, e.g., human Eag1, or Eag2 orthologs, areadded to the assay so as to compete for binding of the antisera to theimmobilized antigen. The ability of the added proteins to compete forbinding of the antisera to the immobilized protein is compared to theability of the hEag2 encoded by SEQ ID NO:2 to compete with itself. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith each of the added proteins listed above are selected and pooled.The cross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

[0177] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein, thought to be perhaps an allele or polymorphic variant of Eag2,to the immunogen protein. In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the protein encoded by Eag2 that is required to inhibit50% of binding, then the second protein is said to specifically bind tothe polyclonal antibodies generated to the respective Eag2 immunogen.

[0178] Other Assay Formats

[0179] Western blot (immunoblot) analysis is used to detect and quantifythe presence of the Eag2 in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind Eag2. The anti-Eag2 antibodies specifically bindto Eag2 on the solid support. These antibodies may be directly labeledor alternatively may be subsequently detected using labeled antibodies(e.g., labeled sheep anti-mouse antibodies) that specifically bind tothe anti-Eag2 antibodies.

[0180] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41.(1986)).

[0181] Reduction of Non-Specific Binding

[0182] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays.Particularly, where the assay involves an antigen or antibodyimmobilized on a solid substrate it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this technique involves coating the substrate with aproteinaceous composition. In particular, protein compositions such asbovine serum albumin (BSA), nonfat powdered milk, and gelatin are widelyused with powdered milk being most preferred.

[0183] Labels

[0184] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0185] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0186] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. The ligands andtheir targets can be used in any suitable combination with antibodiesthat recognize Eag2, or secondary antibodies that recognize anti-Eag2antibodies.

[0187] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0188] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0189] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0190] VI. Assays for Modulators of Eag2

[0191] A. Assays

[0192] Eag2 monomers and Eag2 alleles and polymorphic variants aresubunits of potassium channels. The activity of a potassium channelcomprising Eag2 can be assessed using a variety of in vitro and in vivoassays, e.g., measuring current, measuring membrane potential, measuringion flux, e.g., potassium or rubidium, measuring ligand binding,measuring potassium concentration, measuring second messengers andtranscription levels, using potassium-dependent yeast growth assays, andusing e.g., voltage-sensitive dyes, radioactive tracers, and patch-clampelectrophysiology.

[0193] Furthermore, such assays can be used to test for inhibitors andactivators of channels comprising Eag2. Such modulators of a potassiumchannel are useful for treating various disorders involving potassiumchannels. Treatment of dysfunctions include, e.g., CNS disorders such asmigraines, hearing and vision problems, Alzheimer's disease, learningand memory disorders, Alzheimer's disease, learning and memorydisorders, seizures, psychotic disorders, and use as neuroprotectiveagents (e.g., to prevent stroke). Such modulators are also useful forinvestigation of the channel diversity provided by Eag2 and theregulation/modulation of potassium channel activity provided by Eag2.

[0194] Modulators of the potassium channels are tested usingbiologically active Eag2, either recombinant or naturally occurring.Human Eag2 can be isolated, co-expressed or expressed in a cell, orexpressed in a membrane derived from a cell. In such assays, Eag2 isexpressed alone to form a homomeric potassium channel or is co-expressedwith a second alpha subunit (e.g., another Eag family member, preferablyan Eag subfamily member) so as to form a heteromeric potassium channel.Eag2 can also be expressed with additional beta subunits. Modulation istested using one of the in vitro or in vivo assays described above.Samples or assays that are treated with a potential potassium channelinhibitor or activator are compared to control samples without the testcompound, to examine the extent of modulation. Control samples(untreated with activators or inhibitors) are assigned a relativepotassium channel activity value of 100. Inhibition of channelscomprising Eag2 is achieved when the potassium channel activity valuerelative to the control is about 90%, preferably 50%, more preferably25%. Activation of channels comprising Eag2 is achieved when thepotassium channel activity value relative to the control is 110%, morepreferably 150%, more preferable 200% higher. Compounds that increasethe flux of ions will cause a detectable increase in the ion currentdensity by increasing the probability of a channel comprising Eag2 beingopen, by decreasing the probability of it being closed, by increasingconductance through the channel, and/or by allowing the passage of ions.

[0195] Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell gr membraneexpressing the potassium channel comprising Eag2. A preferred means todetermine changes in cellular polarization is by measuring changes incurrent (thereby measuring changes in polarization) with voltage-clampand patch-clamp techniques, e.g., the “cell-attached” mode, the“inside-out” mode, and the “whole cell” mode (see, e.g., Ackerman etal., New Engl. J. Med. 336:1575-1595 (1997)). Whole cell currents areconveniently determined using the standard methodology (see, e.g., Hamilet al., PFlugers. Archiv. 391:85 (1981). Other known assays include:radiolabeled rubidium flux assays and fluorescence assays usingion-sensitive dyes, voltage-sensitive dyes (see, e.g.,Vestergarrd-Bogind et al., J. Membrane Biol. 88:67-75 (1988); Daniel etal., J. Pharmacol. Meth. 25:185-193 (1991); Holevinsky et al., J.Membrane Biology 137:59-70 (1994)). Assays for compounds capable ofinhibiting or increasing potassium flux through the channel proteinscomprising Eag2 can be performed by application of the compounds to abath solution in contact with and comprising cells having a channel ofthe present invention (see, e.g., Blatz et al., Nature 323:718-720(1986); Park, J. Physiol. 481:555-570 (1994)). Generally, the compoundsto be tested are present in the range from 1 pM to 100 mM.

[0196] The effects of the test compounds upon the function of thechannels can be measured by changes in the electrical currents or ionicflux or by the consequences of changes in currents and flux. Changes inelectrical current or ionic flux are measured by either increases ordecreases in flux of ions such as potassium or rubidium ions. Thecations can be measured in a variety of standard ways. They can bemeasured directly by concentration changes of the ions or indirectly bymembrane potential or by radio-labeling of the ions. Consequences of thetest compound on ion flux can be quite varied. Accordingly, any suitablephysiological change can be used to assess the influence of a testcompound on the channels of this invention. The effects of a testcompound can be measured by a toxin binding assay. When the functionalconsequences are determined using intact cells or animals, one can alsomeasure a variety of effects such as transmitter release (e.g.,dopamine), hormone release (e.g., insulin), transcriptional changes toboth known and uncharacterized genetic markers (e.g., northern blots),cell volume changes (e.g., in red blood cells), immunoresponses (e.g., Tcell activation), changes in cell metabolism such as cell growth or pHchanges, and changes in intracellular second messengers such as Ca²⁺, orcyclic nucleotides.

[0197] Preferably, the Eag2 that is a part of the potassium channel usedin the assay will have at least 85% identity to the amino acid sequencedisplayed in SEQ ID NO:2 or a conservatively modified variant thereofAlternatively, the Eag2 of the assay will be derived from a eukaryoteand include an amino acid subsequence having substantial amino acidsequence identity to the C-terminal region of hEag2. Generally, theamino acid sequence identity will be at least 70%, preferably at least75, 80, 85 or 90%, most preferably at least 95%.

[0198] Human Eag2 orthologs will generally confer substantially similarproperties on a channel comprising such Eag2, as described above. In apreferred embodiment, the cell placed in contact with a compound that issuspected to be a Eag2 homolog is assayed for increasing or decreasingion flux in a eukaryotic cell, e.g., an oocyte of Xenopus (e.g., Xenopuslaevis) or a mammalian cell such as a CHO or HeLa cell. Channels thatare affected by compounds in ways similar to hEag2 are consideredhomologs or orthologs of hEag2.

[0199] B. Modulators

[0200] The compounds tested as modulators of Eag2 channels comprising ahuman Eag2 subunit can be any small chemical compound, or a biologicalentity, such as a protein, sugar, nucleic acid or lipid. Alternatively,modulators can be genetically altered versions of a human Eag2 subunit.Typically, test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

[0201] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0202] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0203] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

[0204] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0205] C. Solid State and soluble high throughput assays

[0206] In one embodiment the invention provides soluble assays usingpotassium channels comprising an Eag2 polypeptide, e.g., hEag2; amembrane comprising an Eag2 potassium channel, or a cell or tissueexpressing potassium channels comprising a Eag2 polypeptide, eithernaturally occurring or recombinant. In another embodiment, the inventionprovides solid phase based in vitro assays in a high throughput format,where Eag2 potassium channel, or a cell or cell membrane expressing anEag2 potassium channel, is attached to a solid phase substrate.

[0207] In the high throughput assays of the invention, it is possible toscreen up to several thousand different modulators or ligands in asingle day. In particular, each well of a microtiter plate can be usedto run a separate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100- about 1500different compounds. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds are possible using the integrated systems of theinvention.

[0208] The channel of interest, or a cell or membrane comprising thechannel of interest can be bound to the solid state component, directlyor indirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest (e.g., the taste transduction molecule of interest)is attached to the solid support by interaction of the tag and the tagbinder.

[0209] A number of tags and tag binders can be used, based upon knownmolecular interactions well described in the literature. For example,where a tag has a natural binder, for example, biotin, protein A, orprotein G, it can be used in conjunction with appropriate tag binders(avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin,etc.) Antibodies to molecules with natural binders such as biotin arealso widely available and appropriate tag binders+; see, SIGMAImmunochemicals 1998 catalogue SIGMA, St. Louis Mo.).

[0210] Similarly, any haptenic or antigenic compound can be used incombination with an appropriate antibody to form a tag/tag binder pair.Thousands of specific antibodies are commercially available and manyadditional antibodies are described in the literature. For example, inone common configuration, the tag is a first antibody and the tag binderis a second antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

[0211] Synthetic polymers, such as polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, and polyacetates can also form anappropriate tag or tag binder. Many other tag/tag binder pairs are alsouseful in assay systems described herein, as would be apparent to one ofskill upon review of this disclosure.

[0212] Common linkers such as peptides, polyethers, and the like canalso serve as tags, and include polypeptide sequences, such as poly glysequences of between about 5 and 200 amino acids. Such flexible linkersare known to persons of skill in the art. For example, poly(ethelyneglycol) linkers are available from Shearwater Polymers, Inc. Huntsville,Ala. These linkers optionally have amide linkages, sulfhydryl linkages,or heterofunctional linkages.

[0213] Tag binders are fixed to solid substrates using any of a varietyof methods currently available. Solid substrates are commonlyderivatized or functionalized by exposing all or a portion of thesubstrate to a chemical reagent which fixes a chemical group to thesurface which is reactive with a portion of the tag binder. For example,groups which are suitable for attachment to a longer chain portion wouldinclude amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanesand hydroxyalkylsilanes can be used to functionalize a variety ofsurfaces, such as glass surfaces. The construction of such solid phasebiopolymer arrays is well described in the literature. See, e.g.,Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solidphase synthesis of, e.g., peptides); Geysen et al., J. Immun. Meth.102:259-274 (1987) (describing synthesis of solid phase components onpins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describingsynthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759(1996) (all describing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

[0214] VII. Computer Assisted Drug Design Using Eag2

[0215] Yet another assay for compounds that modulate the activities ofEag2 involves computer assisted drug design, in which a computer systemis used to generate a three-dimensional structure of Eag2 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands or otherpotassium channel subunits. These regions are then used to identifyligands that bind to the protein or region where Eag2 interacts withother potassium channel subunits.

[0216] The three-dimensional structural model of the protein isgenerated by entering channel protein amino acid sequences of at least50, preferably 75, 100, or 150 amino acid residues or correspondingnucleic acid sequences encoding an Eag2 monomer into the computersystem. The amino acid sequence of each of the monomers is selected fromthe group consisting of SEQ ID NO:2 and a conservatively modifiedversions thereof. The amino acid sequence represents the primarysequence or subsequence of each of the proteins, which encodes thestructural information of the protein. At least 50, preferably 75, 100,or 150 residues of the amino acid sequence (or a nucleotide sequenceencoding at least 50, preferably 75, 100, or 150 amino acids) areentered into the computer system from computer keyboards, computerreadable substrates that include, but are not limited to, electronicstorage media (e.g., magnetic diskettes, tapes, cartridges, and chips),optical media (e.g., CD ROM), information distributed by interrietsites, and by RAM. The three-dimensional structural model of the channelprotein is then generated by the interaction of the amino acid sequenceand the computer system, using software known to those of skill in theart. The resulting three-dimensional computer model can then be saved ona computer readable substrate.

[0217] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary andquaternary structure of the monomer and the heteromeric potassiumchannel protein comprising four monomers. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” oranisotropic terms and primarily include electrostatic potentials,hydrophobic potentials, solvent accessible surfaces, and hydrogenbonding. Secondary energy terms include van der Waals potentials.Biological molecules form the structures that minimize the energy termsin a cumulative fashion. The computer program is therefore using theseterms encoded by the primary structure or amino acid sequence to createthe secondary structural model.

[0218] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0219] Once the structure has been generated, potential ligand bindingregions are identified by the computer system. Three-dimensionalstructures for potential ligands are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential ligand is thencompared to that of Eag2 protein to identify ligands that bind to Eag2.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein.

[0220] Computer systems are also used to screen for mutations,polymorphic variants, alleles, and interspecies homologs of Eag2 genes.Such mutations can be associated with disease states. Once the variantsare identified, diagnostic assays can be used to identify patientshaving such mutated genes associated with disease states. Identificationof the mutated Eag2 genes involves receiving input of a first nucleicacid, e.g., SEQ ID NO:1, or an amino acid sequence encoding Eag2,selected from the group consisting of SEQ ID NO:2, and a conservativelymodified versions thereof. The sequence is entered into the computersystem as described above. The first nucleic acid or amino acid sequenceis then compared to a second nucleic acid or amino acid sequence thathas substantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in Eag2 genes, and mutations associatedwith disease states. The first and second sequences described above canbe saved on a computer readable substrate.

[0221] Human Eag2 monomers and the potassium channels containing theseEag2 monomers can be used with high density oligonucleotide arraytechnology (e.g., GeneChip™) to identify homologs and polymorphicvariants of Eag2 in this invention. In the case where the homologs beingidentified are linked to a known disease, they can be used withGeneChip™ as a diagnostic tool in detecting the disease in a biologicalsample, see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14:869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al.,Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat. Biotechnol.14:1675-1680 (1996); Gingerias et al., Genome Res. 8:435-448 (1998);Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

[0222] VIII. Cellular Transfection and Gene Therapy

[0223] The present invention provides the nucleic acids of Eag2 for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid for Eag2, under thecontrol of a promoter, then expresses a Eag2 monomer of the presentinvention, thereby mitigating the effects of absent, partialinactivation, or abnormal expression of the Eag2 gene.

[0224] Such gene therapy procedures have been used to correct acquiredand inherited genetic defects, cancer, and viral infection in a numberof contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies (for a review of gene therapy procedures, see Anderson,Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993);Mitani & Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology (Doerfler & Böhmeds., 1995); and Yu et al., Gene Therapy 1: 13-26 (1994)).

[0225] Delivery of the gene or genetic material into the cell is thefirst critical step in gene therapy treatment of disease. A large numberof delivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell.

[0226] Methods of non-viral delivery of nucleic acids includelipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in, e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787;and U.S. Pat. No. 4,897,355 and lipofection reagents are soldcommercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutrallipids that are suitable for efficient receptor-recognition lipofectionof polynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration).

[0227] The preparation of lipid:nucleic acid complexes, includingtargeted liposomes such as immunolipid complexes, is well known to oneof skill in the art (see, e.g., Crystal, Science 270:404-410 (1995);Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem.5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad etal., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183,4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,4,837,028, and 4,946,787).

[0228] The use of RNA or DNA viral based systems for the delivery ofnucleic acids take advantage of highly evolved processes for targeting avirus to specific cells in the body and trafficking the viral payload tothe nucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

[0229] The tropism of a retrovirus can be altered by incorporatingforeign envelope proteins, expanding the potential target population oftarget cells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV),and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

[0230] In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. US.A. 81:6466-6470 (1984); and Samulskiet al., J. Virol. 63:03822-3828 (1989).

[0231] In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

[0232] pLASN and MFG-S are examples are retroviral vectors that havebeen used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995);Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl.Acad. Sci. U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the firsttherapeutic vector used in a gene therapy trial. (Blaese et al., Science270:475-480 (1995)). Transduction efficiencies of 50% or greater havebeen observed for MFG-S packaged vectors (Ellem et al., ImmunolImmunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2(1997)).

[0233] Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 Virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

[0234] Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 241:5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

[0235] In many gene therapy applications, it is desirable that the genetherapy vector be delivered with a high degree of specificity to aparticular tissue type. A viral vector is typically modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the viruses outer surface. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. US.A. 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other pairs of virus expressing a ligand fusion protein and targetcell expressing a receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences thought to favor uptakeby specific target cells.

[0236] Gene therapy vectors can be delivered in vivo by administrationto an individual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

[0237] Ex vivo cell transfection for diagnostics, research, or for genetherapy (e.g., via re-infusion of the transfected cells into the hostorganism) is well known to those of skill in the art. In a preferredembodiment, cells are isolated from the subject organism, transfectedwith a nucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

[0238] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.)containing therapeutic nucleic acids can be also administered directlyto the organism for transduction of cells in vivo. Alternatively, nakedDNA can be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

[0239] Administration is by any of the routes normally used forintroducing a molecule into ultimate contact with blood or tissue cells.The nucleic acids are administered in any suitable manner, preferablywith pharmaceutically acceptable carriers. Suitable methods ofadministering such nucleic acids are available and well known to thoseof skill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

[0240] IX. Pharmaceutical Compositions

[0241] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered (e.g., nucleic acid,protein, modulatory compounds or transduced cell), as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed., 1989).

[0242] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the packaged nucleicacid suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, microcrystallinecellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate,stearic acid, and other excipients, colorants, fillers, binders,diluents, buffering agents, moistening agents, preservatives, flavoringagents, dyes, disintegrating agents, and pharmaceutically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,e.g., sucrose, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

[0243] The compound of choice, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

[0244] Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

[0245] Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

[0246] The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. Such doses are administered prophylactically or to anindividual already suffering from the disease. The compositions areadministered to a patient in an amount sufficient to elicit an effectiveprotective or therapeutic response in the patient. An amount adequate toaccomplish this is defined as “therapeutically effective dose.” The dosewill be determined by the efficacy of the particular Eag2 modulators(e.g., Eag2 antagonists and anti-eag2 antibodies) employed and thecondition of the subject, as well as the body weight or surface area ofthe area to be treated. The size of the dose also will be determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular compound or vector in aparticular subject.

[0247] In determining the effective amount of the compound to beadministered in the treatment or prophylaxis of conditions owing todiminished or aberrant expression of the Eag2 channels comprising ahuman Eag2 alpha subunit, the physician evaluates circulating plasmalevels of the compound, compound toxicities, progression of the disease,and the production of antibodies. In general, the dose equivalent of acompound is from about 1 μg to 100 μg for a typical 70 kilogram patient.

[0248] For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

[0249] Transduced cells are prepared for reinfusion according toestablished methods (see, e.g., Abrahamsen et al., J. Clin. Apheresis6:48—53 (1991); Carter et al., J. Clin. Apheresis 4:113-117 (1998);Aebersold et al., J. Immunol. Meth. 112:1-7 (1998); Muul et al, J.Immunol. Methods 101:171-181 (1987); and Carter et al., Transfusion27:362-365 (1987)).

[0250] X. Kits

[0251] Human Eag2 and its homologs are useful tools for examiningexpression and regulation of potassium channels. Human Eag2-specificreagents that specifically hybridize to Eag2 nucleic acid, such as Eag2probes and primers, and Eag2-specific reagents that specifically bind tothe Eag2 protein, e.g., Eag2 antibodies are used to examine expressionand regulation.

[0252] Nucleic acid assays for the presence of Eag2 DNA and RNA in asample include numerous techniques are known to those skilled in theart, such as Southern analysis, northern analysis, dot blots, RNaseprotection, S1 analysis, amplification techniques such as PCR and LCR,and in situ hybridization. In in situ hybridization, for example, thetarget nucleic acid is liberated from its cellular surroundings in suchas to be available for hybridization within the cell while preservingthe cellular morphology for subsequent interpretation and analysis. Thefollowing articles provide an overview of the art of in situhybridization: Singer et al, Biotechniques 4:230-250 (1986); Haase etal, Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic AcidHybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, Eag2 protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant Eag2monomers) and a negative control.

[0253] The present invention also provides for kits for screeningmodulators of the heteromeric potassium channels. Such kits can beprepared from readily available materials and reagents. For example,such kits can comprise any one or more of the following materials: Eag2monomers, reaction tubes, and instructions for testing the activities ofpotassium channels containing Eag2. A wide variety of kits andcomponents can be prepared according to the present invention, dependingupon the intended user of the kit and the particular needs of the user.For example, the kit can be tailored for in vitro or in vivo assays formeasuring the activity of a potassium channel comprising a Eag2 monomer.

[0254] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0255] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

[0256] The following example is provided by way of illustration only andnot by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

Example I Cloning and Expression of hEAG2

[0257] Using PCR and primers, according to standard conditions, hEag2was amplified from human brain tissue cDNA, e.g., whole brain orhippocampus cDNA. The following primers were used for amplification:ATGCCGGGGGGCAAGAGAGGGCTG; (SEQ ID NO:3) CTGACCCTAAGCTCATAAGGATGAAC; (SEQID NO:4) CCACCTCATCATCCTGGATGACTTCC; (SEQ ID NO:5)TTAAAAGTGGATTTCATCTTTGTCAGATTCAGG; (SEQ ID NO:6)GGGGACCTCATTTACCATGCTGGAG (SEQ ID NO:7) GATTCCCTCATCCACATTTTCAAAGGC.(SEQ ID NO:8)

[0258] The SEQ ID NO:3 and 6 oligos also include additional adaptorsequences used for expression vector construction (enzyme sites, Kozakconsensus) that are not present in Eag2 (additional adaptor sequencesnot shown). SEQ ID NO:7 can be used with SEQ ID NO:6 to amplify a regionof Eag2 extending from the putative cyclic nucleotide binding domainthrough the stop codon. SEQ ID NO:8 can be used with SEQ ID NO:3 toamplify a region extending from the initiator methionine to the S4domain. SEQ ID NOS:3 and 6 were used to amplify the entire codingregion. SEQ ID NOS:4 and 5 can be used to amplify an approximately 950bp fragment from just upstream of S3 to the putative cyclic nucleotidebinding domain. SEQ ID NOS:3 and 5 can be used to amplify from theinitiator methionine to the cyclic-nucleotide binding domain (thesequence encoding approximately amino acids 1-720), and SEQ ID NOS:4 and6 can be used to amplify from S3 to the stop codon. At least one ofthese primers should amplify all splice variants. The cDNA was preparedfrom total mRNA isolated from human brain tissue, e.g., whole brain orhippocampus, according to standard methods. hEag2 was amplified with theprimers described above using the following conditions: 30 seconds at95° C., 15 seconds at 68-58° C., and 3 minutes at 72° C. for 40 cycles.

[0259] The PCR products were subcloned into plasmids and sequencedaccording to standard techniques. The nucleotide and amino acidsequences of hEag2 are provided, respectively, in SEQ ID NO:1 and SEQ IDNO:2 (see also FIG. 1).

[0260] mRNA distribution of hEag2 was examined according to standardtechniques. In a northern blot, the Eag2 probe recognized anapproximately 12 kb transcript in the brain, and also fainter 8 and 4.5kb transcripts in the brain. In a dot blot, Eag2 expression was thehighest in the occipital lobe, thalamus, temporal lobe, nucleusaccumbens and pituitary gland. Expression was also detected in frontallobe, hippocampus, cerebral cortex, medulla, adrenal gland, and testis.

Example II Expression and Voltage-Gated Activity of Homomeric ChannelsContaining hEag2 Monomers

[0261] hEag2 monomer was expressed in both Xenopus oocytes and CHO cellsaccording to standard methodology, to demonstrate its ability to formhomomeric potassium channels with voltage-gated activity. Changes incurrent magnitude can be indirectly measured using a reportervoltage-sensitive fluorescent dye (see; e.g., Etts et al., Chemistry andPhysiology of Lipids, 69:137 (1994)). Changes in current magnitude canalso be measured directly using electrophysiology, and by measuring ionflux.

[0262] hEag2 channels are potassium selective and voltage-sensitive.When Eag2 was functionally expressed in either Xenopus oocytes ormammalian cells, it produced an outwardly rectifying potassium currentwith relatively weak voltage-dependence (see FIGS. 2 and 3). Activationof the Eag2 current begins at subthreshold potentials, with the midpointof the activation curve lying between approximately −30 and −20 mV.There is no evidence of inactivation. These properties potentially givethe Eag2 current a role in contributing to cellular resting potentialsand influencing excitability in both the subthreshold and suprathresholdvoltage ranges (that means that the Eag2 current could influence thewhether or not a neuron fires and action potential and/or the shape ofthat action potential). The activation rate of Eag2 is very sensitive toholding potential. If a depolarization occurs from a very hyperpolarizedpotential, Eag2 activation is very slow, occurring over several seconds.However, activation potentials close to typical cell resting potentials(−80 to −50 mV) is very rapid, allowing the Eag2 outward current tocounteract the depolarization quickly.

1 9 1 2967 DNA Homo sapiens CDS (1)..(2967) human ether a go-go (Eag) 2voltage-gated potassium channel 1 atg ccg ggg ggc aag aga ggg ctg gtggca ccg cag aac aca ttt ttg 48 Met Pro Gly Gly Lys Arg Gly Leu Val AlaPro Gln Asn Thr Phe Leu 1 5 10 15 gag aac atc gtc agg cgc tcc agt gaatca agt ttc tta ctg gga aat 96 Glu Asn Ile Val Arg Arg Ser Ser Glu SerSer Phe Leu Leu Gly Asn 20 25 30 gcc cag att gtg gat tgg cct gta gtt tatagt aat gac ggt ttt tgt 144 Ala Gln Ile Val Asp Trp Pro Val Val Tyr SerAsn Asp Gly Phe Cys 35 40 45 aaa ctc tct gga tat cat cga gct gac gtc atgcag aaa agc agc act 192 Lys Leu Ser Gly Tyr His Arg Ala Asp Val Met GlnLys Ser Ser Thr 50 55 60 tgc agt ttt atg tat ggg gaa ttg act gac aag aagacc att gag aaa 240 Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys Lys ThrIle Glu Lys 65 70 75 80 gtc agg caa act ttt gac aac tac gaa tca aac tgcttt gaa gtt ctt 288 Val Arg Gln Thr Phe Asp Asn Tyr Glu Ser Asn Cys PheGlu Val Leu 85 90 95 ctg tac aag aaa aac aga acc cct gtt tgg ttt tat atgcaa att gca 336 Leu Tyr Lys Lys Asn Arg Thr Pro Val Trp Phe Tyr Met GlnIle Ala 100 105 110 cca ata aga aat gaa cat gaa aag gtg gtc ttg ttc ctgtgt act ttc 384 Pro Ile Arg Asn Glu His Glu Lys Val Val Leu Phe Leu CysThr Phe 115 120 125 aag gat att acg ttg ttc aaa cag cca ata gag gat gattca aca aaa 432 Lys Asp Ile Thr Leu Phe Lys Gln Pro Ile Glu Asp Asp SerThr Lys 130 135 140 ggt tgg acg aaa ttt gcc cga ttg aca cgg gct ttg acaaat agc cga 480 Gly Trp Thr Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr AsnSer Arg 145 150 155 160 agt gtt ttg cag cag ctc acg cca atg aat aaa acagag gtg gtc cat 528 Ser Val Leu Gln Gln Leu Thr Pro Met Asn Lys Thr GluVal Val His 165 170 175 aaa cat tca aga cta gct gaa gtt ctt cag ctg ggatca gat atc ctt 576 Lys His Ser Arg Leu Ala Glu Val Leu Gln Leu Gly SerAsp Ile Leu 180 185 190 cct cag tat aaa caa gaa gcg cca aag acg cca ccacac att att tta 624 Pro Gln Tyr Lys Gln Glu Ala Pro Lys Thr Pro Pro HisIle Ile Leu 195 200 205 cat tat tgt gct ttt aaa act act tgg gat tgg gtgatt tta att ctt 672 His Tyr Cys Ala Phe Lys Thr Thr Trp Asp Trp Val IleLeu Ile Leu 210 215 220 acc ttc tac acc gcc att atg gtt cct tat aat gtttcc ttc aaa aca 720 Thr Phe Tyr Thr Ala Ile Met Val Pro Tyr Asn Val SerPhe Lys Thr 225 230 235 240 aag cag aac aac ata gcc tgg ctg gta ctg gatagt gtg gtg gac gtt 768 Lys Gln Asn Asn Ile Ala Trp Leu Val Leu Asp SerVal Val Asp Val 245 250 255 att ttt ctg gtt gac atc gtt tta aat ttt cacacg act ttc gtg ggg 816 Ile Phe Leu Val Asp Ile Val Leu Asn Phe His ThrThr Phe Val Gly 260 265 270 ccc ggt gga gag gtc att tct gac cct aag ctcata agg atg aac tat 864 Pro Gly Gly Glu Val Ile Ser Asp Pro Lys Leu IleArg Met Asn Tyr 275 280 285 ctg aaa act tgg ttt gtg atc gat ctg ctg tcttgt tta cct tat gac 912 Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser CysLeu Pro Tyr Asp 290 295 300 atc atc aat gcc ttt gaa aat gtg gat gag ggaatc agc agt ctc ttc 960 Ile Ile Asn Ala Phe Glu Asn Val Asp Glu Gly IleSer Ser Leu Phe 305 310 315 320 agt tct tta aaa gtg gtg cgt ctc tta cgactg ggc cgt gtg gct agg 1008 Ser Ser Leu Lys Val Val Arg Leu Leu Arg LeuGly Arg Val Ala Arg 325 330 335 aaa ctg gac cat tac cta gaa tat gga gcagca gtc ctc gtg ctc ctg 1056 Lys Leu Asp His Tyr Leu Glu Tyr Gly Ala AlaVal Leu Val Leu Leu 340 345 350 gtg tgt gtg ttt gga ctg gtg gcc cac tggctg gcc tgc ata tgg tat 1104 Val Cys Val Phe Gly Leu Val Ala His Trp LeuAla Cys Ile Trp Tyr 355 360 365 agc atc gga gac tac gag gtc att gat gaagtc act aac acc atc caa 1152 Ser Ile Gly Asp Tyr Glu Val Ile Asp Glu ValThr Asn Thr Ile Gln 370 375 380 ata gac agt tgg ctc tac cag ctg gct ttgagc att ggg act cca tat 1200 Ile Asp Ser Trp Leu Tyr Gln Leu Ala Leu SerIle Gly Thr Pro Tyr 385 390 395 400 cgc tac aat acc agt gct ggg ata tgggaa gga gga ccc agc aag gat 1248 Arg Tyr Asn Thr Ser Ala Gly Ile Trp GluGly Gly Pro Ser Lys Asp 405 410 415 tca ttg tac gtg tcc tct ctc tac tttacc atg aca agc ctt aca acc 1296 Ser Leu Tyr Val Ser Ser Leu Tyr Phe ThrMet Thr Ser Leu Thr Thr 420 425 430 ata gga ttt gga aac ata gct cct accaca gat gtg gag aag atg ttt 1344 Ile Gly Phe Gly Asn Ile Ala Pro Thr ThrAsp Val Glu Lys Met Phe 435 440 445 tcg gtg gct atg atg atg gtt ggc tctctt ctt tat gca act att ttt 1392 Ser Val Ala Met Met Met Val Gly Ser LeuLeu Tyr Ala Thr Ile Phe 450 455 460 gga aat gtt aca aca att ttc cag caaatg tat gcc aac acc aac cga 1440 Gly Asn Val Thr Thr Ile Phe Gln Gln MetTyr Ala Asn Thr Asn Arg 465 470 475 480 tac cat gag atg ctg aat aat gtacgg gac ttc cta aaa ctc tat cag 1488 Tyr His Glu Met Leu Asn Asn Val ArgAsp Phe Leu Lys Leu Tyr Gln 485 490 495 gtc cca aaa ggc ctt agt gag cgagtc atg gat tat att gtc tca aca 1536 Val Pro Lys Gly Leu Ser Glu Arg ValMet Asp Tyr Ile Val Ser Thr 500 505 510 tgg tcc atg tca aaa ggc att gataca gaa aag gtc ctc tcc atc tgt 1584 Trp Ser Met Ser Lys Gly Ile Asp ThrGlu Lys Val Leu Ser Ile Cys 515 520 525 ccc aag gac atg aga gct gat atctgt gtt cat cta aac cgg aag gtt 1632 Pro Lys Asp Met Arg Ala Asp Ile CysVal His Leu Asn Arg Lys Val 530 535 540 ttt aat gaa cat cct gct ttt cgattg gcc agc gat ggg tgt ctg cgc 1680 Phe Asn Glu His Pro Ala Phe Arg LeuAla Ser Asp Gly Cys Leu Arg 545 550 555 560 gcc ttg gcg gta gag ttc caaacc att cac tgt gct ccc ggg gac ctc 1728 Ala Leu Ala Val Glu Phe Gln ThrIle His Cys Ala Pro Gly Asp Leu 565 570 575 att tac cat gct gga gaa agtgtg gat gcc ctc tgc ttt gtg gtg tca 1776 Ile Tyr His Ala Gly Glu Ser ValAsp Ala Leu Cys Phe Val Val Ser 580 585 590 gga tcc ttg gaa gtc atc caggat gat gag gtg gtg gct att tta ggg 1824 Gly Ser Leu Glu Val Ile Gln AspAsp Glu Val Val Ala Ile Leu Gly 595 600 605 aag ggt gat gta ttt gga gacatc ttc tgg aag gaa acc acc ctt gcc 1872 Lys Gly Asp Val Phe Gly Asp IlePhe Trp Lys Glu Thr Thr Leu Ala 610 615 620 cat gca tgt gcg aac gtc cgggca ctg acg tac tgt gac cta cac atc 1920 His Ala Cys Ala Asn Val Arg AlaLeu Thr Tyr Cys Asp Leu His Ile 625 630 635 640 atc aag cgg gaa gcc ttgctc aaa gtc ctg gac ttt tat aca gct ttt 1968 Ile Lys Arg Glu Ala Leu LeuLys Val Leu Asp Phe Tyr Thr Ala Phe 645 650 655 gca aac tcc ttc tca aggaat ctc act ctt act tgc aat ctg agg aaa 2016 Ala Asn Ser Phe Ser Arg AsnLeu Thr Leu Thr Cys Asn Leu Arg Lys 660 665 670 cgg atc atc ttt cgt aagatc agt gat gtg aag aaa gag gag gag gag 2064 Arg Ile Ile Phe Arg Lys IleSer Asp Val Lys Lys Glu Glu Glu Glu 675 680 685 cgc ctc cgg cag aag aatgag gtg acc ctc agc att ccc gtg gac cac 2112 Arg Leu Arg Gln Lys Asn GluVal Thr Leu Ser Ile Pro Val Asp His 690 695 700 cca gtc aga aag ctc ttccag aag ttc aag cag cag aag gag ctg cgg 2160 Pro Val Arg Lys Leu Phe GlnLys Phe Lys Gln Gln Lys Glu Leu Arg 705 710 715 720 aat cag ggc tca acacag ggt gac cct gag agg aac caa ctc cag gta 2208 Asn Gln Gly Ser Thr GlnGly Asp Pro Glu Arg Asn Gln Leu Gln Val 725 730 735 gag agc cgc tcc ttacag aat gga acc tcc atc acc gga acc agc gtg 2256 Glu Ser Arg Ser Leu GlnAsn Gly Thr Ser Ile Thr Gly Thr Ser Val 740 745 750 gtg act gtg tca cagatt act ccc att cag acg tct ctg gcc tat gtg 2304 Val Thr Val Ser Gln IleThr Pro Ile Gln Thr Ser Leu Ala Tyr Val 755 760 765 aaa acc agt gaa tccctt aag cag aac aac cgt gat gcc atg gaa ctc 2352 Lys Thr Ser Glu Ser LeuLys Gln Asn Asn Arg Asp Ala Met Glu Leu 770 775 780 aag ccc aac ggc ggtgct gac caa aaa tgt ctc aaa gtc aac agc cca 2400 Lys Pro Asn Gly Gly AlaAsp Gln Lys Cys Leu Lys Val Asn Ser Pro 785 790 795 800 ata aga atg aagaat gga aat gga aaa ggg tgg ctg cga ctc aag aat 2448 Ile Arg Met Lys AsnGly Asn Gly Lys Gly Trp Leu Arg Leu Lys Asn 805 810 815 aat atg gga gcccat gag gag aaa aag gaa gac tgg aat aat gtc act 2496 Asn Met Gly Ala HisGlu Glu Lys Lys Glu Asp Trp Asn Asn Val Thr 820 825 830 aaa gct gag tcaatg ggg cta ttg tct gag gac ccc aag agc agt gat 2544 Lys Ala Glu Ser MetGly Leu Leu Ser Glu Asp Pro Lys Ser Ser Asp 835 840 845 tca gag aac agtgtg acc aaa aac cca cta agg aaa aca gat tct tgt 2592 Ser Glu Asn Ser ValThr Lys Asn Pro Leu Arg Lys Thr Asp Ser Cys 850 855 860 gac agt gga attaca aaa agt gac ctt cgt ttg gat aag gct ggg gag 2640 Asp Ser Gly Ile ThrLys Ser Asp Leu Arg Leu Asp Lys Ala Gly Glu 865 870 875 880 gcc cga agtccg cta gag cac agt ccc atc cag gct gat gcc aag cac 2688 Ala Arg Ser ProLeu Glu His Ser Pro Ile Gln Ala Asp Ala Lys His 885 890 895 ccc ttt tatccc atc ccc gag cag gcc tta cag acc aca ctg cag gaa 2736 Pro Phe Tyr ProIle Pro Glu Gln Ala Leu Gln Thr Thr Leu Gln Glu 900 905 910 gtc aaa cacgaa ctc aaa gag gac atc cag ctg ctc agc tgc aga atg 2784 Val Lys His GluLeu Lys Glu Asp Ile Gln Leu Leu Ser Cys Arg Met 915 920 925 act gcc ctagaa aag cag gtg gca gaa att tta aaa ata ctg tcg gaa 2832 Thr Ala Leu GluLys Gln Val Ala Glu Ile Leu Lys Ile Leu Ser Glu 930 935 940 aaa agc gtaccc cag gcc tca tct ccc aaa tcc caa atg cca ctc caa 2880 Lys Ser Val ProGln Ala Ser Ser Pro Lys Ser Gln Met Pro Leu Gln 945 950 955 960 gta cccccc cag ata cca tgt cag gat att ttt agt gtc tca agg cct 2928 Val Pro ProGln Ile Pro Cys Gln Asp Ile Phe Ser Val Ser Arg Pro 965 970 975 gaa tcacct gaa tct gac aaa gat gaa atc cac ttt taa 2967 Glu Ser Pro Glu Ser AspLys Asp Glu Ile His Phe 980 985 2 988 PRT Homo sapiens 2 Met Pro Gly GlyLys Arg Gly Leu Val Ala Pro Gln Asn Thr Phe Leu 1 5 10 15 Glu Asn IleVal Arg Arg Ser Ser Glu Ser Ser Phe Leu Leu Gly Asn 20 25 30 Ala Gln IleVal Asp Trp Pro Val Val Tyr Ser Asn Asp Gly Phe Cys 35 40 45 Lys Leu SerGly Tyr His Arg Ala Asp Val Met Gln Lys Ser Ser Thr 50 55 60 Cys Ser PheMet Tyr Gly Glu Leu Thr Asp Lys Lys Thr Ile Glu Lys 65 70 75 80 Val ArgGln Thr Phe Asp Asn Tyr Glu Ser Asn Cys Phe Glu Val Leu 85 90 95 Leu TyrLys Lys Asn Arg Thr Pro Val Trp Phe Tyr Met Gln Ile Ala 100 105 110 ProIle Arg Asn Glu His Glu Lys Val Val Leu Phe Leu Cys Thr Phe 115 120 125Lys Asp Ile Thr Leu Phe Lys Gln Pro Ile Glu Asp Asp Ser Thr Lys 130 135140 Gly Trp Thr Lys Phe Ala Arg Leu Thr Arg Ala Leu Thr Asn Ser Arg 145150 155 160 Ser Val Leu Gln Gln Leu Thr Pro Met Asn Lys Thr Glu Val ValHis 165 170 175 Lys His Ser Arg Leu Ala Glu Val Leu Gln Leu Gly Ser AspIle Leu 180 185 190 Pro Gln Tyr Lys Gln Glu Ala Pro Lys Thr Pro Pro HisIle Ile Leu 195 200 205 His Tyr Cys Ala Phe Lys Thr Thr Trp Asp Trp ValIle Leu Ile Leu 210 215 220 Thr Phe Tyr Thr Ala Ile Met Val Pro Tyr AsnVal Ser Phe Lys Thr 225 230 235 240 Lys Gln Asn Asn Ile Ala Trp Leu ValLeu Asp Ser Val Val Asp Val 245 250 255 Ile Phe Leu Val Asp Ile Val LeuAsn Phe His Thr Thr Phe Val Gly 260 265 270 Pro Gly Gly Glu Val Ile SerAsp Pro Lys Leu Ile Arg Met Asn Tyr 275 280 285 Leu Lys Thr Trp Phe ValIle Asp Leu Leu Ser Cys Leu Pro Tyr Asp 290 295 300 Ile Ile Asn Ala PheGlu Asn Val Asp Glu Gly Ile Ser Ser Leu Phe 305 310 315 320 Ser Ser LeuLys Val Val Arg Leu Leu Arg Leu Gly Arg Val Ala Arg 325 330 335 Lys LeuAsp His Tyr Leu Glu Tyr Gly Ala Ala Val Leu Val Leu Leu 340 345 350 ValCys Val Phe Gly Leu Val Ala His Trp Leu Ala Cys Ile Trp Tyr 355 360 365Ser Ile Gly Asp Tyr Glu Val Ile Asp Glu Val Thr Asn Thr Ile Gln 370 375380 Ile Asp Ser Trp Leu Tyr Gln Leu Ala Leu Ser Ile Gly Thr Pro Tyr 385390 395 400 Arg Tyr Asn Thr Ser Ala Gly Ile Trp Glu Gly Gly Pro Ser LysAsp 405 410 415 Ser Leu Tyr Val Ser Ser Leu Tyr Phe Thr Met Thr Ser LeuThr Thr 420 425 430 Ile Gly Phe Gly Asn Ile Ala Pro Thr Thr Asp Val GluLys Met Phe 435 440 445 Ser Val Ala Met Met Met Val Gly Ser Leu Leu TyrAla Thr Ile Phe 450 455 460 Gly Asn Val Thr Thr Ile Phe Gln Gln Met TyrAla Asn Thr Asn Arg 465 470 475 480 Tyr His Glu Met Leu Asn Asn Val ArgAsp Phe Leu Lys Leu Tyr Gln 485 490 495 Val Pro Lys Gly Leu Ser Glu ArgVal Met Asp Tyr Ile Val Ser Thr 500 505 510 Trp Ser Met Ser Lys Gly IleAsp Thr Glu Lys Val Leu Ser Ile Cys 515 520 525 Pro Lys Asp Met Arg AlaAsp Ile Cys Val His Leu Asn Arg Lys Val 530 535 540 Phe Asn Glu His ProAla Phe Arg Leu Ala Ser Asp Gly Cys Leu Arg 545 550 555 560 Ala Leu AlaVal Glu Phe Gln Thr Ile His Cys Ala Pro Gly Asp Leu 565 570 575 Ile TyrHis Ala Gly Glu Ser Val Asp Ala Leu Cys Phe Val Val Ser 580 585 590 GlySer Leu Glu Val Ile Gln Asp Asp Glu Val Val Ala Ile Leu Gly 595 600 605Lys Gly Asp Val Phe Gly Asp Ile Phe Trp Lys Glu Thr Thr Leu Ala 610 615620 His Ala Cys Ala Asn Val Arg Ala Leu Thr Tyr Cys Asp Leu His Ile 625630 635 640 Ile Lys Arg Glu Ala Leu Leu Lys Val Leu Asp Phe Tyr Thr AlaPhe 645 650 655 Ala Asn Ser Phe Ser Arg Asn Leu Thr Leu Thr Cys Asn LeuArg Lys 660 665 670 Arg Ile Ile Phe Arg Lys Ile Ser Asp Val Lys Lys GluGlu Glu Glu 675 680 685 Arg Leu Arg Gln Lys Asn Glu Val Thr Leu Ser IlePro Val Asp His 690 695 700 Pro Val Arg Lys Leu Phe Gln Lys Phe Lys GlnGln Lys Glu Leu Arg 705 710 715 720 Asn Gln Gly Ser Thr Gln Gly Asp ProGlu Arg Asn Gln Leu Gln Val 725 730 735 Glu Ser Arg Ser Leu Gln Asn GlyThr Ser Ile Thr Gly Thr Ser Val 740 745 750 Val Thr Val Ser Gln Ile ThrPro Ile Gln Thr Ser Leu Ala Tyr Val 755 760 765 Lys Thr Ser Glu Ser LeuLys Gln Asn Asn Arg Asp Ala Met Glu Leu 770 775 780 Lys Pro Asn Gly GlyAla Asp Gln Lys Cys Leu Lys Val Asn Ser Pro 785 790 795 800 Ile Arg MetLys Asn Gly Asn Gly Lys Gly Trp Leu Arg Leu Lys Asn 805 810 815 Asn MetGly Ala His Glu Glu Lys Lys Glu Asp Trp Asn Asn Val Thr 820 825 830 LysAla Glu Ser Met Gly Leu Leu Ser Glu Asp Pro Lys Ser Ser Asp 835 840 845Ser Glu Asn Ser Val Thr Lys Asn Pro Leu Arg Lys Thr Asp Ser Cys 850 855860 Asp Ser Gly Ile Thr Lys Ser Asp Leu Arg Leu Asp Lys Ala Gly Glu 865870 875 880 Ala Arg Ser Pro Leu Glu His Ser Pro Ile Gln Ala Asp Ala LysHis 885 890 895 Pro Phe Tyr Pro Ile Pro Glu Gln Ala Leu Gln Thr Thr LeuGln Glu 900 905 910 Val Lys His Glu Leu Lys Glu Asp Ile Gln Leu Leu SerCys Arg Met 915 920 925 Thr Ala Leu Glu Lys Gln Val Ala Glu Ile Leu LysIle Leu Ser Glu 930 935 940 Lys Ser Val Pro Gln Ala Ser Ser Pro Lys SerGln Met Pro Leu Gln 945 950 955 960 Val Pro Pro Gln Ile Pro Cys Gln AspIle Phe Ser Val Ser Arg Pro 965 970 975 Glu Ser Pro Glu Ser Asp Lys AspGlu Ile His Phe 980 985 3 24 DNA Artificial Sequence Description ofArtificial SequencePCR amplification primer 3 atgccggggg gcaagagagg gctg24 4 26 DNA Artificial Sequence Description of Artificial SequencePCRamplification primer 4 ctgaccctaa gctcataagg atgaac 26 5 26 DNAArtificial Sequence Description of Artificial SequencePCR amplificationprimer 5 ccacctcatc atcctggatg acttcc 26 6 33 DNA Artificial SequenceDescription of Artificial SequencePCR amplification primer 6 ttaaaagtggatttcatctt tgtcagattc agg 33 7 25 DNA Artificial Sequence Description ofArtificial SequencePCR amplification primer 7 ggggacctca tttaccatgctggag 25 8 27 DNA Artificial Sequence Description of ArtificialSequencePCR amplification primer 8 gattccctca tccacatttt caaaggc 27 9962 PRT Homo sapiens human ether a go-go (Eag) 1 voltage-gated potassiumchannel 9 Met Thr Met Ala Gly Gly Arg Arg Gly Leu Val Ala Pro Gln AsnThr 1 5 10 15 Phe Leu Glu Asn Ile Val Arg Arg Ser Asn Asp Thr Asn PheVal Leu 20 25 30 Gly Asn Ala Gln Ile Val Asp Trp Pro Ile Val Tyr Ser AsnAsp Gly 35 40 45 Phe Cys Lys Leu Ser Gly Tyr His Arg Ala Glu Val Met GlnLys Ser 50 55 60 Ser Thr Cys Ser Phe Met Tyr Gly Glu Leu Thr Asp Lys AspThr Ile 65 70 75 80 Glu Lys Val Arg Gln Thr Phe Glu Asn Tyr Glu Met AsnSer Phe Glu 85 90 95 Ile Leu Met Tyr Lys Lys Asn Arg Thr Pro Val Trp PhePhe Val Lys 100 105 110 Ile Ala Pro Ile Arg Asn Glu Gln Asp Lys Val ValLeu Phe Leu Cys 115 120 125 Thr Phe Ser Asp Ile Thr Ala Phe Lys Gln ProIle Glu Asp Asp Ser 130 135 140 Cys Lys Gly Trp Gly Lys Phe Ala Arg LeuThr Arg Ala Leu Thr Ser 145 150 155 160 Ser Arg Ser Val Leu Gln Gln LeuAla Pro Ser Val Gln Lys Gly Glu 165 170 175 Asn Val His Lys His Ser ArgLeu Ala Glu Val Leu Gln Leu Gly Ser 180 185 190 Asp Ile Leu Pro Gln TyrLys Gln Glu Ala Pro Lys Thr Pro Pro His 195 200 205 Ile Ile Leu His TyrCys Val Phe Lys Thr Thr Trp Asp Trp Ile Ile 210 215 220 Leu Ile Leu ThrPhe Tyr Thr Ala Ile Leu Val Pro Tyr Asn Val Ser 225 230 235 240 Phe LysThr Arg Gln Asn Asn Val Ala Trp Leu Val Val Asp Ser Ile 245 250 255 ValAsp Val Ile Phe Leu Val Asp Ile Val Leu Asn Phe His Thr Thr 260 265 270Phe Val Gly Pro Ala Gly Glu Val Ile Ser Asp Pro Lys Leu Ile Arg 275 280285 Met Asn Tyr Leu Lys Thr Trp Phe Val Ile Asp Leu Leu Ser Cys Leu 290295 300 Pro Tyr Asp Val Ile Asn Ala Phe Glu Asn Val Asp Glu Gly Ile Ser305 310 315 320 Ser Leu Phe Ser Ser Leu Lys Val Val Arg Leu Leu Arg LeuGly Arg 325 330 335 Val Ala Arg Lys Leu Asp His Tyr Ile Glu Tyr Gly AlaAla Val Leu 340 345 350 Val Leu Leu Val Cys Val Phe Gly Leu Ala Ala HisTrp Met Ala Cys 355 360 365 Ile Trp Tyr Ser Ile Gly Asp Tyr Glu Ile PheAsp Glu Asp Thr Lys 370 375 380 Thr Ile Arg Asn Asn Ser Trp Leu Tyr GlnLeu Ala Met Asp Ile Gly 385 390 395 400 Thr Pro Tyr Gln Phe Asn Gly SerGly Ser Gly Lys Trp Glu Gly Gly 405 410 415 Pro Ser Lys Asn Ser Val TyrIle Ser Ser Leu Tyr Phe Thr Met Thr 420 425 430 Ser Leu Thr Ser Val GlyPhe Gly Asn Ile Ala Pro Ser Thr Asp Ile 435 440 445 Glu Lys Ile Phe AlaVal Ala Ile Met Met Ile Gly Ser Leu Leu Tyr 450 455 460 Ala Thr Ile PheGly Asn Val Thr Thr Ile Phe Gln Gln Met Tyr Ala 465 470 475 480 Asn ThrAsn Arg Tyr His Glu Met Leu Asn Ser Val Arg Asp Phe Leu 485 490 495 LysLeu Tyr Gln Val Pro Lys Gly Leu Ser Glu Arg Val Met Asp Tyr 500 505 510Ile Val Ser Thr Trp Ser Met Ser Arg Gly Ile Asp Thr Glu Lys Val 515 520525 Leu Gln Ile Cys Pro Lys Asp Met Arg Ala Asp Ile Cys Val His Leu 530535 540 Asn Arg Lys Val Phe Lys Glu His Pro Ala Phe Arg Leu Ala Ser Asp545 550 555 560 Gly Cys Leu Arg Ala Leu Ala Met Glu Phe Gln Thr Val HisCys Ala 565 570 575 Pro Gly Asp Leu Ile Tyr His Ala Gly Glu Ser Val AspSer Leu Cys 580 585 590 Phe Val Val Ser Gly Ser Leu Glu Val Ile Gln AspAsp Glu Val Val 595 600 605 Ala Ile Leu Gly Lys Gly Asp Val Phe Gly AspVal Phe Trp Lys Glu 610 615 620 Ala Thr Leu Ala Gln Ser Cys Ala Asn ValArg Ala Leu Thr Tyr Cys 625 630 635 640 Asp Leu His Val Ile Lys Arg AspAla Leu Gln Lys Val Leu Glu Phe 645 650 655 Tyr Thr Ala Phe Ser His SerPhe Ser Arg Asn Leu Ile Leu Thr Tyr 660 665 670 Asn Leu Arg Lys Arg IleVal Phe Arg Lys Ile Ser Asp Val Lys Arg 675 680 685 Glu Glu Glu Glu ArgMet Lys Arg Lys Asn Glu Ala Pro Leu Ile Leu 690 695 700 Pro Pro Asp HisPro Val Arg Arg Leu Phe Gln Arg Phe Arg Gln Gln 705 710 715 720 Lys GluAla Arg Leu Ala Ala Glu Arg Gly Gly Arg Asp Leu Asp Asp 725 730 735 LeuAsp Val Glu Lys Gly Asn Val Leu Thr Glu His Ala Ser Ala Asn 740 745 750His Ser Leu Val Lys Ala Ser Val Val Thr Val Arg Glu Ser Pro Ala 755 760765 Thr Pro Val Ser Phe Gln Ala Ala Ser Thr Ser Gly Val Pro Asp His 770775 780 Ala Lys Leu Gln Ala Pro Gly Ser Glu Cys Leu Gly Pro Lys Gly Gly785 790 795 800 Gly Gly Asp Cys Ala Lys Arg Lys Ser Trp Ala Arg Phe LysAsp Ala 805 810 815 Cys Gly Lys Ser Glu Asp Trp Asn Lys Val Ser Lys AlaGlu Ser Met 820 825 830 Glu Thr Leu Pro Glu Arg Thr Lys Ala Ser Gly GluAla Thr Leu Lys 835 840 845 Lys Thr Asp Ser Cys Asp Ser Gly Ile Thr LysSer Asp Leu Arg Leu 850 855 860 Asp Asn Val Gly Glu Ala Arg Ser Pro GlnAsp Arg Ser Pro Ile Leu 865 870 875 880 Ala Glu Val Lys His Ser Phe TyrPro Ile Pro Glu Gln Thr Leu Gln 885 890 895 Ala Thr Val Leu Glu Val ArgHis Glu Leu Lys Glu Asp Ile Lys Ala 900 905 910 Leu Asn Ala Lys Met ThrAsn Ile Glu Lys Gln Leu Ser Glu Ile Leu 915 920 925 Arg Ile Leu Thr SerArg Arg Ser Ser Gln Ser Pro Gln Glu Leu Phe 930 935 940 Glu Ile Ser ArgPro Gln Ser Pro Glu Ser Glu Arg Asp Ile Phe Gly 945 950 955 960 Ala Ser

What is claimed is:
 1. An isolated nucleic acid encoding an alphasubunit of a potassium channel, wherein the subunit: (i) forms, with atleast one additional Eag family alpha subunit, a potassium channelhaving the characteristic of voltage sensitivity; and (ii) comprises anamino acid sequence that has greater than 70% identity to amino acids720-988 of a human Eag2 amino acid sequence.
 2. An isolated nucleic acidencoding an alpha subunit of a potassium channel, wherein the subunit:(i) forms, with at least one additional Eag family alpha subunit, apotassium channel having the characteristic of voltage sensitivity; and(ii) comprises an amino acid sequence that has greater than 85% identityto the amino acid sequence of SEQ ID NO:2.
 3. The isolated nucleic acidof claim 1, wherein the polypeptide specifically binds to polyclonalantibodies generated against SEQ ID NO:2
 4. The isolated nucleic acid ofclaim 1, wherein the nucleic acid encodes human Eag2.
 5. The isolatednucleic acid of claim 1, wherein the nucleic acid encodes an amino acidsequence of SEQ ID NO:2.
 6. The isolated nucleic acid sequence of claim1, wherein the nucleic acid has a nucleotide sequence of SEQ ID NO: 1.7. The isolated nucleic acid of claim 1, wherein the nucleic acid isamplified by primers that selectively hybridize under stringenthybridization conditions to the same sequence as primers selected fromthe group consisting of: ATGCCGGGGGGCAAGAGAGGGCTG (SEQ ID NO:3);CTGACCCTAAGCTCATAAGGATGAAC (SEQ ID NO:4); CCACCTCATCATCCTGGATGACTTCC(SEQ ID NO:5), TTAAAAGTGGATTTCATCTTTGTCAGATTCAGG (SEQ ID NO:6);GGGGACCTCATTTACCATGCTGGAG (SEQ ID NO:7); and GATTCCCTCATCCACATTTTCAAAGGC(SEQ ID NO:8).
 8. The isolated nucleic acid of claim 1, wherein thepolypeptide monomer comprises an alpha subunit of a homomeric channel.9. The isolated nucleic acid of claim 1, wherein the polypeptide monomercomprises an alpha subunit of a heteromeric channel.
 10. An expressionvector comprising the nucleic acid of claim
 1. 11. A host celltransfected with the vector of claim
 10. 12. An isolated nucleic acid ofclaim 1, which nucleic acid selectively hybridizes under moderatelystringent hybridization conditions to a nucleotide sequence of SEQ IDNO:1.
 13. An isolated nucleic acid encoding an Eag2 polypeptide, thenucleic acid specifically hybridizing under stringent conditions to anucleotide sequence of SEQ ID NO:1.
 14. An isolated nucleic acidencoding an Eag2 polypeptide, wherein the isolated nucleic acidspecifically hybridizes to a nucleic acid encoding an amino acid of SEQID NO:2.
 15. A method of detecting a nucleic acid, the method comprisingcontacting the nucleic acid with an isolated nucleic acid of claim 1.16. An isolated alpha subunit of a potassium channel, wherein thesubunit: (i) forms, with at least one additional Eag family alphasubunit, a potassium channel having a characteristic of voltagesensitivity; and (ii) comprises an amino acid sequence that has greaterthan 70% amino acid identity to amino acids 720-988 of a human Eag2amino acid sequence.
 17. An isolated alpha subunit of a potassiumchannel, wherein the subunit: (i) forms, with at least one additionalEag family alpha subunit, a potassium channel having a characteristic ofvoltage sensitivity; and (ii) comprises an amino acid sequence that hasgreater than 85% amino acid identity to the amino acid sequence of SEQID NO:2.
 18. The isolated alpha subunit of claim 16, wherein thepolypeptide specifically binds to polyclonal antibodies generatedagainst SEQ ID NO:2.
 19. The isolated alpha subunit of claim 16, whereinthe polypeptide has a molecular weight of between about 109 kD and about119 kD.
 20. The isolated alpha subunit of claim 16, wherein thepolypeptide monomer has an amino acid sequence of human Eag2.
 21. Theisolated polypeptide monomer of claim 16, wherein the polypeptidemonomer has an amino acid sequence of SEQ ID NO:2.
 22. The isolatedpolypeptide monomer of claim 16, wherein the polypeptide monomercomprises an alpha subunit of a homomeric potassium channel.
 23. Theisolated polypeptide monomer of claim 16, wherein the polypeptidemonomer comprises an alpha subunit of a heteromeric potassium channel.24. An antibody that selectively binds to the alpha subunit of claim 16.25. An antibody of claim 24, wherein the alpha subunit has an amino acidsequence of SEQ ID NO:2.
 26. A method for identifying a compound thatincreases or decreases ion flux through an Eag potassium channel, themethod comprising the steps of: (i) contacting the compound an alphasubunit of a potassium channel, wherein the subunit: (a) forms, with atleast one additional Eag family alpha subunit, a potassium channelhaving a characteristic of voltage sensitivity; and (b) comprises anamino acid sequence that has greater than 70% identity to amino acids720-988 of a human Eag2 amino acid sequence; and (ii) determining thefunctional effect of the compound upon the potassium channel.
 27. Amethod for identifying a compound that increases or decreases ion fluxthrough an Eag potassium channel, the method comprising the steps of:(i) contacting the compound an alpha subunit of a potassium channel,wherein the subunit: (a) forms, with at least one additional Eag familyalpha subunit, a potassium channel having a characteristic of voltagesensitivity; and (b) comprises an amino acid sequence that has greaterthan 85% identity to an amino acid sequence of SEQ ID NO:2; and (ii)determining the functional effect of the compound upon the potassiumchannel.
 28. The method of claim 26, wherein the functional effect is aphysical effect.
 29. The method of claim 26, wherein the functionaleffect is a chemical effect.
 30. The method of claim 26, wherein thefunctional effect is determined by measuring changes in ion flux, ionconcentration, current, or voltage.
 31. The method of claim 26, whereinthe functional effect is determined by measuring ligand binding to thechannel.
 32. The method of claim 26, wherein the polypeptide is attachedto a solid support.
 33. The method of claim 26, wherein the alphasubunit is recombinant.
 34. The method of claim 26, wherein thepotassium channel is homomeric.
 35. The method of claim 26, wherein thepotassium channel is heteromeric.
 36. The method of claim 26, whereinthe alpha subunit has an amino acid sequence of SEQ ID NO:2.
 37. Themethod of claim 26, wherein the polypeptide is expressed in a host cellor cell membrane.
 38. The method of claim 37, wherein the cell is aeukaryotic cell.
 39. The method of claim 37, wherein the cell is a humancell.
 40. A method for identifying a compound that increases ordecreases ion flux through a potassium channel comprising an Eag2polypeptide, the method comprising the steps of: (i) entering into acomputer system an amino acid sequence of at least 50 amino acids of anEag2 polypeptide or at least 150 nucleotides of a nucleic acid encodingthe Eag2 polypeptide, the Eag2 polypeptide comprising a subsequencehaving at least 70% amino acid sequence identity to amino acids 720 to988 of SEQ ID NO:2 or at least 85% amino acid sequence identity to anamino acid sequence of SEQ ID NO:2; (ii) generating a three-dimensionalstructure of the polypeptide encoded by the amino acid sequence; (iii)generating a three-dimensional structure of the potassium channelcomprising the Eag2 polypeptide; (iv) generating a three-dimensionalstructure of the compound; and (v) comparing the three-dimensionalstructures of the polypeptide and the compound to determine whether ornot the compound binds to the polypeptide.
 41. A method of modulatingion flux through an Eag potassium channel comprising an Eag2 polypeptideto treat disease in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of acompound identified using the method of claim 26 or
 40. 42. A method ofdetecting the presence of human Eag2 in a biological sample, the methodcomprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a human Eag2-specific reagent thatselectively associates with human Eag2; and, (iii) detecting the levelof human Eag2-specific reagent that selectively associates with thesample.
 43. The method of claim 42, wherein the human Eag 2-specificreagent is selected from the group consisting of: Eag 2-specificantibodies, Eag 2-specific oligonucleotide primers, and Eag 2-specificnucleic acid probes.
 44. In a computer system, a method of screening formutations of a human Eag2 gene, the method comprising the steps of: (i)entering into the system at least about 50 nucleotides of a firstnucleic acid sequence encoding a human Eag2 gene having a nucleotidesequence of SEQ ID NO: 1, and conservatively modified variants thereof;(ii) comparing the first nucleic acid sequence with a second nucleicacid sequence having substantial identity to the first nucleic acidsequence; and (iii) identifying nucleotide differences between the firstand second nucleic acid sequences.
 45. The method of claim 44, whereinthe second nucleic acid sequence is associated with a disease state.